US6342583B1 - cDNAs coding for members of the carcinoembryonic antigen family - Google Patents

cDNAs coding for members of the carcinoembryonic antigen family Download PDF

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US6342583B1
US6342583B1 US08/027,974 US2797493A US6342583B1 US 6342583 B1 US6342583 B1 US 6342583B1 US 2797493 A US2797493 A US 2797493A US 6342583 B1 US6342583 B1 US 6342583B1
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Thomas R. Barnett
James J. Elting
Michael E. Kamarck
Axel W. Kretschmer
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Siemens Healthcare Diagnostics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention concerns nucleic acid sequences which code for carcinoembryonic antigen (CEA) antigen family peptide sequences.
  • CEA carcinoembryonic antigen
  • CEA Carcinoembryonic antigen was first described by Gold and Freedman, J. Exp. Med., 121, 439-462, (1965).
  • CEA is characterized as a glycoprotein of approximately 200,000 molecular weight with 50-60% by weight of carbohydrate. CEA is present during normal human fetal development, but only in very low concentration in the normal adult intestinal tract. It is produced and secreted by a number of different tumors.
  • CEA is a clinically useful tumor marker for the management of colorectal cancer patients.
  • CEA can be measured using sensitive immunoassay methods.
  • presurgical serum levels of CEA When presurgical serum levels of CEA are elevated, a postsurgical drop in serum CEA to the normal range typically indicates successful resection of the tumor.
  • Postsurgical CEA levels that do not return to normal often indicate incomplete resection of the tumor or the presence of additional tumor sites in the patient. After returning to normal levels, subsequent rapid rises in serum CEA levels usually indicate the presence of metastages. Slower postsurgical rises from the normal level are most often interpreted to indicate the presence of new primary tumors not previously detected. Post surgical management of colon cancer patients is thus facilitated by the measurement of CEA.
  • CEA is a member of an antigen family. Because of this, the immunoassay of CEA by presently available methods is complicated by the fact that CEA is but one of several potentially reactive antigens. There have been at least sixteen CEA-like antigens described in the literature. Since some of these appear to be the same antigen described by different investigators, the actual number of different antigens is somewhat less than this number. Nonetheless, there is a complex array of cross-reactive antigens which can potentially interfere with an immunoassay of the CEA released by tumors. It is known that serum levels of CEA-like antigens are elevated in many non-cancerous conditions such an inflammatory liver diseases and also in smokers.
  • the members of the “CEA family” share some antigenic determinants. These common epitopes are not useful in distinguishing the members of the antigen family and antibodies recognizing them are of little use for measuring tumor-specific CEA levels.
  • CEA is a mixture of antigens (components A and B in this case).
  • U.S. Pat. No. 3,697,638 mentions methods for separating and radioiodinating each component and their use in specific RIA's.
  • U.S. Pat. No. 3,852,415 entitled “Compositions for Use in Radioimmunoassay, as Substitute for Blood Plasma Extract in Determination of Carcinoembryonic Antigen” relates to the use of a buffer containing EDTA and bovine serum albumin as a substitute for plasma as a diluent for CEA RIA's.
  • U.S. Pat. No. 4,228,236, entitled “Process of Producing Carcinoembryonic Antigen”, is directed to the use of the established cell lines LS-174T and LS-180 or clones or derivatives thereof for the production of CEA.
  • U.S. Pat. No. 4,272,504 entitled “Antibody Adsorbed Support Method for Carcinoembryonic Antigen Assay”, concerns two concepts for the radioimmunoassay of CEA.
  • U.S. Pat. No. 4,272,504 relates to a sample pretreatment in the form of heating to 65 to 85° C. at pH 5 to precipitate and eliminate extraneous protein.
  • U.S. Pat. No. 4,299,815, entitled “Carcinoembryonic Antigen Determination”, concerns diluting a CEA sample with water and pretreating by heating to a temperature below which precipitation of protein will occur. The pretreated sample is then immunoassayed using RIA, EIA, FIA or chemiluminescent immunoassay.
  • U.S. Pat. No. 4,467,031 entitled “Enzyme-Immunoassay for Carcinoembryonic Antigen”, relates to a sandwich enzyme immunoassay for CEA in which the first of two anti-CEA monoclonal antibodies is attached to a solid phase and the second monoclonal is conjugated with peroxidase.
  • EP 113072-A entitled “Assaying Blood Sample for Carcinoembryonic Antigen—After Removal of Interfering Materials by Incubation with Silica Gel”, relates to the removal from a serum of a plasma sample of interfering substances by pretreatment with silica gel. The precleared sample is then subjected to an immunoassay.
  • EP 102008-A entitled “Cancer Diagnostics Carcinoembryonic Antigen—Produced from Perchloric Acid Extracts Without Electrophoresis”, relates to a procedure for the preparation of CEA from perchloric acid extracts, without the use of an electrophoresis step.
  • EP 92223-A entitled “Determination of Carcinoembryonic Antigen in Cytosol or Tissue—for Therapy Control and Early Recognition of Regression”, concerns an immunoassay of CEA, not in serum or plasma, but in the cytosol fraction of the tumor tissue itself.
  • EP 83103759.6 entitled “Cytosole-CEA-Measurement as Predictive Test in Carcinoma, Particularly Mammacarcinoma”, is similar to EP 92223-A.
  • EP 83303759 entitled “Monoclonal Antibodies Specific to Carcinoembryonic Antigen”, relates to the production of “CEA specific” monoclonal antibodies and their use in immunoassays.
  • WO 84/02983 entitled “Specific CEA-Family Antigens, Antibodies Specific Thereto and Their Methods of Use”, is directed to the use of monoclonal antibodies to CEA-meconium (MA)-, and NCA-specific epitopes in immunoassays designed to selectively measure each of these individual components in a sample.
  • MA CEA-meconium
  • NCA-specific epitopes in immunoassays designed to selectively measure each of these individual components in a sample.
  • CEA assays utilize either monoclonal or polyclonal antibodies which are generated by immunizing animals with the intact antigen of choice. None of them address the idea of making sequence specific antibodies for the detection of a unique primary sequence of the various antigens. They do not cover the use of any primary amino acid sequence for the production of antibodies to synthetic peptides or fragments of the natural product. They do not include the concept of using primary amino acid sequences to distinguish the CEA family members. None of them covers the use of DNA or RNA clones for isolating the genes with which to determine the primary sequence.
  • Nucleotide A monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of the pentose) and that combination of base and sugar is called a nucleoside.
  • the base characterizes the nucleotide.
  • the four DNA bases are adenine (“A”), guanine (“G”), cytosine (“C”), and thymine (“T”).
  • the four RNA bases are A, G, C and uracil (“U”).
  • DNA Sequence A linear array of nucleotides connected one to the other by phosphodiester bonds between the 3′ and 5′ carbons of adjacent pentoses.
  • Codon A DNA sequence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a translation start signal or a translation termination signal.
  • the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode the amino acid leucine (“Leu”), TAG, TAA and TGA are translation stop signals and ATG is a translation start signal.
  • Reading Frame The grouping of codons during translation of mRNA into amino acid sequences. During translation, the proper reading frame must be maintained. For example, the sequence GCTGGTTGTAAG may be translated in three reading frames or phases, each of which affords a different amino acid sequence
  • Polypeptide A linear array of amino acids connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids.
  • Genome The entire DNA of a cell or a virus. It includes inter alia the structural genes coding for the polypeptides of the cell or virus, as well as its operator, promoter and ribosome binding and interaction sequences, including sequences such as the Shine-Delgarno sequences.
  • Structural Gene A DNA sequence which encodes through its template or messenger RNA (“mRNA”) a sequence of amino acids characteristics of a specific polypeptide.
  • mRNA messenger RNA
  • Transcription The process of producing mRNA from a structural gene.
  • Translation The process of producing a polypeptide from mRNA.
  • Expression The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
  • Plasmid A non-chromosomal double-stranded DNA sequence comprising an intact “replicon” such that the plasmid is replicated in a host cell.
  • the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid.
  • a plasmid carrying the gene for tetracycline resistance (Tet R ) transforms a cell previously sensitive to tetracycline into one which is resistant to it.
  • a cell transformed by a plasmid is called a “transformant”.
  • Phage or Bacteriophage Bacteriophage—Bacterial virus, many of which consist of DNA sequences encapsulated in a protein envelope or coat (“capsid protein”).
  • Cloning Vehicle A plasmid, phage DNA or other DNA sequence which is capable of replicating in a host cell, which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g., replication, production of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance.
  • a cloning vehicle is often called a vector.
  • Cloning The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.
  • Recombinant DNA Molecule or Hybrid DNA A molecule consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and have the capacity to infect some host cell and be maintained therein.
  • cDNA Expression Vector A procaroytic cloning vehicle which also contains sequences of nucleotides that facilitate expression of cDNA sequences in eucaroytic cells. These nucleotides include sequences that function as eucaryotic promoter, alternative splice sites and polyadenylation signals.
  • Transformation/Transfection DNA or RNA is introduced into cells in such a way as to allow gene expression.
  • “Infected” referred to herein concerns the introduction of RNA or DNA by a viral vector into the host.
  • “Injected” referred to herein concerns the microinjection (use of a small syringe) of DNA into a cell.
  • CEA antigen family (CEA gene family)—a set of genes (gene family) and their products (antigen family) that share nucleotide sequences homologous to partial cDNA LV-7 (CEA -(a)) and as a result of theses similarities also share a subset of their antigenic epitopes.
  • the present invention concerns the following DNA sequences designed as TM-2 (CEA-(e)), TM-3 (CEA-(f)), TM-4 (CEA-(g)), KGCEA1 and KGCEA2, which code for CEA antigen family peptide sequences or nucleic acids having a base sequence (DNA or RNA) that are hybridizable therewith:
  • the present invention is also directed to a replicable recombinant cloning vehicle (“vector”) having an insert comprising a nucleic acid, e.g., DNA, which comprises a base sequence which codes for a CEA peptide or a base sequence hybridizable therewith.
  • a replicable recombinant cloning vehicle having an insert comprising a nucleic acid, e.g., DNA, which comprises a base sequence which codes for a CEA peptide or a base sequence hybridizable therewith.
  • This invention also relates to a cell that is transformed/transfected, infected or injected with the above described replicable recombinant cloning vehicle or nucleic acid hybridizable with the aforementioned cDNA.
  • the invention also concerns the transfection of cells using free nucleic acid, without the use of a cloning vehicle.
  • the present invention concerns a polypeptide expressed by the above described transfected, infected or injected cell, which polypeptide exhibits immunological cross-reactivity with a CEA, as well as labelled forms of the polypeptide.
  • the invention also relates to polypeptides having an amino acid sequence, i.e., synthetic peptides, or the expression product of a cell that is transfected, injected, infected with the above described replicable recombinant cloning vehicles, as well as labelled forms thereof.
  • the present invention concerns a synthetic peptide having an amino acid sequence corresponding to the entire amino acid sequence or a portion thereof having no less than five amino acids of the aforesaid expression product.
  • the invention further relates to an antibody preparation specific for the above described polypeptide.
  • Another aspect of the invention concerns an immunoassay method for detecting CEA or a functional equivalent thereof in a test sample comprising
  • the invention also is directed to a nucleic acid hybridization method for detecting a CEA or a related nucleic acid (DNA or RNA) sample in a test sample comprising
  • nucleic acid probe comprising a nucleic acid, which comprises a base sequence which codes for a CEA peptide sequence or a base sequence that is hybridizable therewith
  • the present invention also concerns a method for detecting the presence of carcinoembryonic antigen or a functional equivalent thereof in an animal or human patient in vivo comprising
  • a labeled e.g., a radio-opaque material that can be detected by X-rays, radiolabeled or labeled with paramagnetic materials that can be detected by NMR
  • a labeled e.g., a radio-opaque material that can be detected by X-rays, radiolabeled or labeled with paramagnetic materials that can be detected by NMR
  • the present invention relates to the use of an antibody preparation according to the present invention for therapeutic purposes, namely, attaching to an antibody preparation radionuclides, toxins or other biological effectors to form a complex and introducing an effective amount of such complex into an animal or human patient, e.g., by injection or orally.
  • the antibody complex would attach to CEA in a patient and the radionuclide, toxin or other biological effector would serve to destroy the CEA expressing cell.
  • FIG. 1 is a schematic representation of the transmembrane CEA's.
  • CEA-(a) partial CEA pcLV7
  • CEA-(b) full coding CEA (pc 15LV7)
  • CEA-(c) TM-1 FL-CEA; pc 19-22)
  • CEA-(d) NCA NCA (pcBT 20) 67711
  • ATCC Nos. 67708, 67709, 67710, 67711 and 67712 were all deposited with the American Type Culture Collection on May 25, 1988.
  • CEA-(a), CEA-(b), CEA-(c) and CEA-(d) are given hereinbelow:
  • FIG. 1 A schematic relationship of the transmembrane CEA's, namely TM-1 (CEA-(c)), TM-2 (CEA-(e)), TM-3 (CEA-(f)) and TM-4 (CEA-(g)) is depicted in FIG. 1 :
  • TM-2 differs from TM-1 in that the 100 amino acids (100 AA) section 14 is deleted and at splice point 20 between sections 12 and 16 , surprisingly an extra amino acid, namely Asp occurs.
  • TM-3 is the same as TM-1 except that section 18 is truncated at splice point 22 , i.e., a section of 70 amino acids is deleted and results in a new section made up of subsections 24 + 26 . Surprisingly, however, six new amino acids (section 26 ) occur.
  • Another example of formation of a novel amino acid sequences relating from a deletion of nucleic acid sequence is for platelet derived growth factor-A.
  • TM-4 is the same as TM-2 up until the end of subsection 24 .
  • Subsection 24 is contained in section 18 of TM-1 and TM-2, but is not depicted in FIG. 1 for TM-1 and TM-2.
  • CEA epitopes are unique. These are the epitopes which have been useful for distinguishing the various CEA-like antigens immunologically.
  • Peptide epitopes are defined by the linear amino acid sequence of the antigen and/or features resulting from protein folding. The information required for protein following is encoded in the primary amino acid sequence. Therefore, antigenic differences ultimately result from differences in the primary structure of the difference CEA molecules. The differences residing in the CEA protein in the CEA species can thus be determined by determining the primary amino acid sequences. This can be most readily accomplished by cloning and sequencing each of the genes for CEA.
  • unique probes can be selected for each gene and expression of each gene can be determined in different tumor types by nucleic acid hybridization techniques.
  • the present invention provides a tool with which to identify potential genes coding for different members of the CEA family and to determine the theoretical primary amino acid sequences for them. Using the method of automated peptide synthesis, peptides can then be synthesized corresponding to unique sequences in these antigens. With these peptides, antibodies to these sequences can be produced which, in the intact CEA molecule, might not be recognized by the animal being immunized. Having accomplished this, advantage can then be taken of the differences in these antigens to generate specific immunoassays for the measurement of each antigen.
  • useful cloning vehicles may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., plasmids from E.
  • coli including col E1, pCR1, pBR322, pMB89 and their derivatives, wider host range plasmids, e.g., RP4, and phage DNAs, e.g., the numerous derivatives of phage, e.g., NM989, and other DNA phages, e.g., M13 and Filêtous single-stranded DNA phages and vectors derived from combinations of plasmids and phage DNAs such as plasmids which have been modified to employ phage DNA or other expression control sequences or yeast plasmids such as the 2 ⁇ plasmids or derivatives thereof.
  • Useful hosts may include bacterial hosts such as strains of E. coli, such as E.
  • each specific cloning vehicle various sites may be selected for insertion of the nucleic acid according to the present invention. These sites are usually designated by the restriction endonuclease which cuts them.
  • the Pst1 site is located in the gene for beta-lactamase, between the nucleotide triplets that code for amino acids 181 and 182 of that protein.
  • One of the two HindII endonuclease recognition sites is between the triplets coding for amino acids 101 and 102 and one of the several Taq sites at the triplet coding for amino acid 45 of beta-lactamase in pBR322.
  • the EcoRI site and the PVUII site in this plasmid lie outside of any coding region, the EcoR1 site being located between the genes coding for resistance to tetracycline and ampicillin, respectively. These sites are well recognized by those of skill in the art. It is, of course, to be understood that a cloning vehicle useful in this invention need not have a restriction endonuclease site for insertion of the chosen DNA fragment. Instead, the vehicle could be cut and joined to the fragment by alternative means.
  • the vector or cloning vehicle and in particular the site chosen therein for attachment of a selected nucleic acid fragment to form a recombinant nucleic acid molecule is determined by a variety of factors, e.g., the number of sites susceptible to a particular restriction enzyme, the size of the protein to be expressed, the susceptibility of the desired protein to proteolytic degradation by host cell enzymes, the contamination of the protein to be expressed by host cell proteins difficult to remove during purification, the expression characteristics, such as the location of start and stop codons relative to the vector sequences, and other factors recognized by those of skill in the art.
  • the choice of a vector and an insertion site for a particular gene is determined by a balance of these factors, not all sections being equally effective for a given case.
  • Methods of inserting nucleic acid sequences into cloning vehicles to form recombinant nucleic acid molecules include, for example, dA-dT tailing, direct ligation, synthetic linkers, exonuclease and polymerase-linked repair reactions followed by ligation, or extension of the nucleic acid strand with an appropriate polymerase and an appropriate single-stranded template followed by ligation.
  • nucleotide sequences or nucleic acid fragments inserted at the selected site of the cloning vehicle may include nucletodies which are not part of the actual structural gene for the desired polypeptide or mature protein or may include only a fragment of the complete structural gene for the desired protein or mature protein.
  • the cloning vehicle or vector containing the foreign gene is employed to transform an appropriate host so as to permit that host to replicate the foreign gene and to express the protein coded by the foreign gene or portion thereof.
  • the selection of an appropriate host is also controlled by a number of factors recognized by the art. These include, for example, the compatibility with the chosen vector, the toxicity of proteins encoded by the hybrid plasmid, the ease of recovery of the desired protein, the expression characteristics, biosafety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for expression of a particular recombinant DNA molecule.
  • the level of production of a protein is governed by two major factors: the number of copies of its gene within the cell and the efficiency with which those gene copies are transcribed and translated. Efficiency of transcription and translation (which together comprise expression) is in turn dependent upon nucleotide sequences, normally situated ahead of the desired coding sequence. These nucleotide sequences or expression control sequences define inter alia, the location at which RNA polymerase interacts to initiate transcription (the promoter sequence) and at which ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation. Not all such expression control sequences function with equal efficiency.
  • the newly engineered nucleic acid, e.g., DNA, fragment may be inserted into a multicopy plasmid or a bacteriophage derivative in order to increase the number of gene copies within the cell and thereby further improve the yield of expressed protein.
  • the gene coding for that polypeptide may be selected and removed from a recombinant nucleic acid molecule containing it and reinserted into a recombinant nucleic acid molecule closer or in a more appropriate relationship to its former expression control sequence or under the control of one of the above described expression control sequences.
  • Such methods are known in the art.
  • relationship may encompass many factors, e.g., the distance separating the expression enhancing and promoting regions of the recombinant nucleic acid molecule and the inserted nucleic acid sequence, the transcription and translation characteristics of the inserted nucleic acid sequence or other sequences in the vector itself, the particular nucleotide sequence of the inserted nucleic acid sequence and other sequences of the vector and the particular characteristics of the expression enhancing and promoting regions of the vector.
  • Particularly useful ⁇ cloning vehicles contain a temperature-sensitive mutation in the repression gene cl and suppressible mutations in gene S, the product of which is necessary for lysis of the host cell, and gene E, the product of which is major capsid protein of the virus.
  • gene S the product of which is necessary for lysis of the host cell
  • gene E the product of which is major capsid protein of the virus.
  • the lysogenic cells are grown at 32° C. and then heated to 45° C. to induce excision of the prophage. Prolonged growth at 37° C. leads to high levels of production of the protein, which is retained within the cells, since these are not lysed by phage gene products in the normal way, and since the phage gene insert is not encapsulated it remains available for further transcription. Artificial lysis of the cells then releases the desired product in high yield.
  • the activity of the polypeptides produced by the recombinant nucleic acid molecules of this invention may be improved by fragmenting, modifying or derivatizing the nucleic acid sequences or polypeptides of this invention by well-known means, without departing from the scope of this invention.
  • polypeptides of the present invention include the following:
  • fragments of polypeptides (1) or (2) above such fragments produced by synthesis of amino acids or by digestion or cleavage.
  • an amount of amino acid equal to six times the molar amount of the growing peptide chain is activated by combining it with one-half as many moles of a carbodiimide (e.g., dicyclohexylcarbodiimide, or diiospropylcarbodiimide) for ten minutes at 0° C., to form the symmetric anhydride of the amino acid.
  • a carbodiimide e.g., dicyclohexylcarbodiimide, or diiospropylcarbodiimide
  • the amino acid used should be provided originally as the N-alpha-tert.-butyloxycarbonyl derivative, with side chains protected with benzyl esters (e.g., aspartic or glutamic acids), benzyl ethers (e.g., serine, threonine, cysteine or tyrosine), benzyloxcarbonyl groups (e.g, lysine) or other protecting groups commonly used in peptide synthesis;
  • benzyl esters e.g., aspartic or glutamic acids
  • benzyl ethers e.g., serine, threonine, cysteine or tyrosine
  • benzyloxcarbonyl groups e.g, lysine
  • the N-alpha-(tert.-butyloxycarbonyl) group is removed from the most recently added amino acid by reacting with 30 to 65%, preferably 50% (v/v) trifluoroacetic acid in methylene chloride for 10 to 30 minutes at room temperature;
  • steps (i) through (h) are repeated until the required peptide sequence has been constructed.
  • the peptide is then removed from the resin and simultaneoulsy the side-chain protecting groups are removed, by reaction with anhydrous hydrofluoric acid containing 10% v/v of anisole or other suitable (aromatic) scavenger. Subsequently, the peptide can be purified by gel filtration, ion exchange, high pressure liquid chromatography, or other suitable means.
  • chemical synthesis can be carried out without the sold phase resin, in which case the synthetic reactions are performed entirely in solution.
  • the reactions are similar and well known in the art, and the final product is essentially identical.
  • Digestion of the polypeptide can be accomplished by using proteolytic enzymes, especially those enzymes whose substrate specificity results in cleavage of the polypeptide at sites immediately adjacent to the desired sequence of amino acids.
  • Cleavage of the polypeptide can be accomplished by chemical means. Particular bonds between amino acids can be cleaved by reaction with specific reagents. Examples include the following: bonds involving methionine are cleaved by cyanogen bromide; asparaginyl-glycine bonds are cleaved by hydroxylamine.
  • the nucleic acids coding for TM-1, TM-2 and TM-3 can be used as probes to isolate other members of the CEA gene family.
  • the nucleic acids coding for TM-1, TM-2 and TM-3 can be used to derive oligonucleotide probes to determine the expression of TM-1, TM-2, TM-3 and other CEA genes in various tumor types.
  • TM-1, TM-2, TM-3 and TM-4 nucleotide sequences can be used to predict the primary amino acid sequence of the protein for production of synthetic peptides.
  • Synthetic peptides derived from the above sequence can be used to produce sequence-specific antibodies.
  • Immunoassays for each member of the CEA antigen family can be produced with these sequence-specific antibodies and synthetic peptides.
  • the aforementioned immunoassays can be used as diagnostics for different types of cancer if it is determined that different members of the CEA family are clinically useful markers for different types of cancer.
  • Peptides according to the present invention can be labelled by conventional means using radioactive moieties (e.g., 125 I), enzymes, dyes and fluorescent compounds, just to name a few.
  • radioactive moieties e.g., 125 I
  • enzymes e.g., enzymes
  • dyes e.g., dyes
  • fluorescent compounds just to name a few.
  • immunoassays can be used.
  • the readout systems capable of being employed in these assays are numerous and non-limiting examples of such systems include fluorescent and colorimetirc enzyme systems, radioisotopic labelling and detection and chemiluminescent systems.
  • Two examples of immunoassay methods are as follows:
  • An enzyme linked immunoassay using an antibody preparation according to the present invention (including Fab or F(ab)′ fragments derived therefrom) to a solid phase (such as a microtiter plate or latex beads) is attached a purified antibody of a specificity other than that which is conjugated to the enzyme.
  • This solid phase antibody is contacted with the sample containing CEA antigen family members. After washing, the solid phase antibody-antigen complex is contacted with the conjugated anti-peptide antibody (or conjugated fragment). After washing away unbound conjugate, color or fluorescence is developed by adding a chromogenic or fluorogenic substrate for the enzyme. The amount of color or fluorescence developed is proportional to the amount of antigen in the sample.
  • the nucleic acid probe is conjugated with a label, for example, an enzyme, a fluorophore, a radioisotope, a chemiluminescent compound, etc.
  • a label for example, an enzyme, a fluorophore, a radioisotope, a chemiluminescent compound, etc.
  • the probed would be contacted with the sample and the presence of any hybridizable nucleic acid sequence would be detected by developing in the presence of a chromogenic enzyme substrate, detection of the fluorophore by epifluorescence, by autoradiography of the radioisotopically labelled probed or by chemiluminescence.
  • the detection of hybridizable RNA sequences can be accomplished by (1) a dot blot methodology or (2) an in sutu hybridization methodology.
  • the invention also relates to the use in medicine of the aforementioned complex of the invention.
  • the invention further provides a pharmaceutical composition containing as an active ingredient a complex of the invention in the form of a sterile and/or physiologically isotonic aqueous solution.
  • solutions and emulsions containing as an active ingredient the complex of the invention should be sterile and, if appropriate, blood-isotonic.
  • the active complex will be administered perorally, parenterally (for example, intramuscularly, intraperitoneally, or intravenously), rectally or locally.
  • RNA was prepared by the proteinase K extraction method of J. Favolaro, R. Treisman and R. Kamen, Methods in Enzymology, 65, 718, Academic Press, Inc. (1980), followed by oligo dT cellulose chromatography to yield poly A+ RNA (3′-polyadenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides).
  • poly A+ RNA 3′-polyadenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides.
  • transfectant 23.411 ATCC No. CRL 9731, deposited with the ATCC on Jun. 1, 1988
  • TM-1, TM-2, TM-3 and TM-4 Kamarck et al, Proc. Natl.
  • cytoplasmic fraction was mixed with an equal volume of 0.2 M tris-HCl, pH 7.8, 23 mM EDTA, 0.3 M NaCl, 2% sodium dodecyl sulfate and 400 ⁇ g/ml of proteinase K, incubated for 1 hour at 37° C., then extracted once with an equal volume of phenol/cholorform (1:1/v:v) solution. Nucleic acids were obtained by ethanol precipitation of the separated aqueous phase. Total RNA was enriched by passage in 0.5 M NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% sarcosyl through an oligo dT(12-18) cellulose column. After washing, bound RNA was eluted in the same solution without sodium chloride.
  • RNA component of the cDNA/mRNA hybrids was replaced with the second complementary strand by treatment with RNase H, E. coli DNA polymerase I and E. coli DNA ligase at 12° C. and 22° C. for 1.5 hours each. Molecular ends were polished by treatment with T4 DNA polymerase.
  • cDNA was phenol/chloroform extracted and purified over a “SEPHADEX G-50” spun column prepared in 10 mM Tris-HCl, ph 7.8, 1 mM EDTA (TE).
  • Ligation proceeded at 7° C. for 2 days in the presence of T4 DNA ligase. Aliquots of the ligation reaction were added to commercially-obtained packaging mix (Stratagene, San Diego, Calif. U.S.A.). Five million phage particles were obtained often in vitro packaging and infection of E. coli host NM514.
  • Phage DNA was prepared according to T. Maniatis, E. Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual , Cold Spring Harbor, (1982). DNA segments were isolated from low melting agarose gels and inserted for subcloning into Bluescript plasmid vectors (Stratagene, San Diego, Calif. U.S.A.). DNA sequencing was performed by the dideoxy termination method of F. Sanger, S. Nicklen and A. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74, 5463-5467, (1977). The nucleic acid and translated sequence for cDNA in pcE22 is given hereinabove (TM-2 (CEA-(e)).
  • RNA was prepared by the proteinase K extraction method of J. Favolaro, R. Treisman and R. Kamen, Methods in Enzymology, 65 718, Academic Press, Inc. (1980), followed by oligo dT cellulose chromatography to yield poly A+ RNA (3′-poladenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides).
  • poly A+ RNA 3′-poladenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides.
  • ATCC HTB38 HT-29 tumor cells
  • Cells were lysed by homogenization in an ice-cold solution of 140 mM NaCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl, pH 8.0, 0.5% NP40, 4 mM dithiothreitol and 20 units of placental ribonuclease inhibitor/ml. Sodium deoxycholate was then added to 0.2%. Cytoplasm and nuclei were separated by centriguation of the homogenate at 12,000 ⁇ g for 20 minutes.
  • cytoplasmic fraction was mixed with an equal volume of 0.2 M tris-Hcl, pH 7.8, 25 mM EDTA, 0.3 M NaCl, 2% sodium dodecyl sulfate and 400 ⁇ g/ml of proteinase K, incubated for 1 hour at 37° C., then extracted once with an equal volume of phenol/choloform (1:1/v:v) solution. Nucleic acids were obtained by ethanol precipitation of the separated aqueous phase. Total RNA was enriched by passage in 0.5 M NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% sarcosyl through an oligo dT(12-18) cellulose column. After washing, bound RNA was eluted in the same solution without sodium chloride.
  • RNA component of the cDNA/mRNA hybrids was replaced with the second complementary strand by treatment with RNase H, E. coli DNA polymerase I and E. coli DNA ligase at 12° C. and 22° C. for 1.5 hours each. Molecular ends were polished by treatment with T4 DNA polymerase.
  • cDNA was phenol/chloroform extracted and purified over a “SEPHADEX G-50” spun column prepared in 10 mM Tris-HCl, pH 7.8, 1 mM EDTA (TE).
  • Phage DNA was prepared according to T. Maniatis, E. Fitsch and J. Sambrook, Molecular Cloning, A Laboratory Manual , Cold Spring Habor, (1982). DNA segments were isolated from low melting agarose gels and inserted for subcloning into Bluescript plasmid vectors (Stratagene, San Diego, Calif., U.S.A.). DNA sequencing was performed by the didexoy termination method of F. Sanger, S. Nicklen and A. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74, 5463-5467, (1977).
  • the nucleic acid and translated sequence for cDNA in HT-6 not complete at the 5′ end of it s coding region, but the nucleotide sequence and restricting map of the HT-6 insert indicates that it is related to nucleic acid sequences of cDNA clones coding for CEA-(c) and CEA-(e).
  • the nucleotide sequence of HT-6 insert differs from these clones at only nucleotide position 1463 to 1515 and 1676 to 2429 of the CEA-(c) cDNA.
  • the pcHT-6 insert is a partial coding sequence for CEA-(f) and the presumed nucleic acid and translated sequence of CEA-(f) is given hereinabove (TM-3 (CEA-(f)).
  • Messenger RNA is prepared by the proteinase K extraction method of J. Favolaro, R. Treisman and R. Kamen, Methos in Enzymology , 65, 718, Academic Press, Inc. (1980), followed by oligo dT cellulose chromatography to yield poly A+ RNA (3′-polyadenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides).
  • poly A+ RNA 3′-polyadenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides.
  • transfectant 23.411 or tumor cell line HT-29 ATCC HTB 38
  • Cells are lysed by homogenization in an ice-cold solution of 140 mM NaCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl, pH 8.0, 0.5% NP40, 4 mM dithiothreitol and 20 units of placental ribonuclease inhibitor/ml. Sodium deoxycholate was then added to 0.2%. Cytoplasm and nuclei are separated by centriguation of the homongenate at 12,000 ⁇ g for 20 minutes.
  • the cytoplasmic fraction is mixed with an equal volume of 0.2 M Tris-Hcl, pH 7.8, 25 mM EDTA, 0.3 M NaCl, 2% sodium dodecyl sulfate and 400 ⁇ g/ml of proteinase K, incubated for 1 hour at 37° C., then extracted once with an equal volume of phenol/cholorform (1:1/v:v) solution. Nucleic acids are obtained by ethanol precipitation of the separated aqueous phase. Total RNA is enriched by passage in 0.5 M NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% sarcosyl through an oligo dT(12-18) cellulose column. After washing, bound RNA is eluted in the same solution without sodium chloride.
  • RNA component of the cDNA/mRNA hybrids is replaced with the second complementary strand by treatment with RNase H, E. coli DNA polymerase I and E. coli DNA ligase at 12° C. and 22° C. for 1.5 hours each.
  • RNase H RNase H
  • E. coli DNA polymerase I E. coli DNA ligase at 12° C. and 22° C. for 1.5 hours each.
  • Molecular ends are polished by treatment with T4 DNA polymerase.
  • cDNA is phenol/cholorform extracted and purified over a “SEPHADEX G-50” spun column prepared in 10 mM Tris-HCl, pH 7.8, 1 mM EDTA (TE).
  • Ligation proceeds at 7° C. for 2 days in the presence of T4 DNA ligase. Aliquots of the ligation reaction are added to commercially-obtained packaging mix (Stratagene, San Diego, Calif., U.S.A.). Phage particles are obtained after in vitro packaging and infection of E. coli host NM514.
  • Phage DNA is prepared according to T. Maniatis, E. Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual , Cold Spring Harbor, (1982). DNA segments are isolated from low melting agarose gels and inserted for subcloning into Bluescript plasmid vectors (Stratagene, San Diego, Calif., U.S.A.). DNA sequencing is performed by the didexoy termination method of F. Sanger, S. Nicklen and A. Coulson, Proc. Natl. Acad. Sci., U.S.A. , 74, 5463-5467, (1977). The nucleotide and translated sequence for a cDNA coding for CEA-(g) is given hereinabove (TM-4 (CEA-(g)).
  • a segment of cDNA coding for a portion of carcinoembroynic antigen [LV7 or CEA-(a)] was radiolabelled by random priming and used to detect homologous sequences on filter replicas of a commercial cDNA library prepared from KG-1 cells in bacteriophage vector ⁇ gt11 (Clontech Laboratories, Inc., Palo Alto, Calif., U.S.A.). Hybridizations were performed at 68° C.
  • cDNA inserts were excised from phage DNA with EcoRI endonuclease and subcloned into the EcoRI site of the plasmid vector pBluescript KS. DNA sequencing on double-stranded DNA was by the method of Sanger et al, supra. The sequences of two different inserts from the KG-1 cDNA library are shown below:
  • pcKGCEA1 1 acagcacagctgacagccgtactcaggaagcttctggatcctaggcttatctccacagag 60 61 gagaacacacacaagcagcagagaccatggggcccctctcagcccctcctgcacacacctc 120 MetGlyProLeuSerAlaProProCysThrHisLeu 121 atcacttggaagggggtcctgctcacagcatcacttttaaacttctggaatccgcccaca 180 IleThrTrpLysGlyValLeuLeuThrAlaSerLeuLeuAsnPheTrpAsnProProThr 181 actgcccaagtcacgattgaagcccagccacccaaagtttctgaggggaaggatgtcttt 240 ThrAlaGl

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Abstract

A nucleic acid comprising a base sequence which codes for a CEA family member peptide sequence or nucleic acids having a base sequence hybridizable therewith, replicable recombinant cloning vehicles having an insert comprising such nucleic acid, cells transfected, infected or injected with such cloning vehicles, polypeptides expressed by such cells, synthetic peptides derived from the coding sequence of CEA family member nucleic acids, antibody preparations specific for such polypeptides, immunoassays for detecting CEA family members using such antibody preparations and nucleic acid hybridization methods for detecting CEA family member nucleic acid sequences using a nucleic acid probe comprising the above described nucleic acid.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS
This is a division of application Ser. No. 07/760,031, filed Sep. 13, 1991, now U.S. Pat. No. 5,231,009, which is a division of Ser. No. 07/274,107 filed Nov. 21, 1988, now U.S. Pat. No. 5,122,599; which is a C-I-P of application Ser. No. 07/207,678 filed Jun. 16, 1988, now abandoned which is a C-I-P of application Ser. No. 07/060,031, filed Jun. 19, 1987, now abandoned, which is a C-I-P of application Ser. No. 07/016,683, filed Feb. 19, 1987, now abandoned, which is a C-I-P of application Ser. No. 06/896,361, filed Aug. 13, 1986, now abandoned.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns nucleic acid sequences which code for carcinoembryonic antigen (CEA) antigen family peptide sequences.
2. Background Information
Carcinoembryonic antigen was first described by Gold and Freedman, J. Exp. Med., 121, 439-462, (1965). CEA is characterized as a glycoprotein of approximately 200,000 molecular weight with 50-60% by weight of carbohydrate. CEA is present during normal human fetal development, but only in very low concentration in the normal adult intestinal tract. It is produced and secreted by a number of different tumors.
CEA is a clinically useful tumor marker for the management of colorectal cancer patients. CEA can be measured using sensitive immunoassay methods. When presurgical serum levels of CEA are elevated, a postsurgical drop in serum CEA to the normal range typically indicates successful resection of the tumor. Postsurgical CEA levels that do not return to normal often indicate incomplete resection of the tumor or the presence of additional tumor sites in the patient. After returning to normal levels, subsequent rapid rises in serum CEA levels usually indicate the presence of metastages. Slower postsurgical rises from the normal level are most often interpreted to indicate the presence of new primary tumors not previously detected. Post surgical management of colon cancer patients is thus facilitated by the measurement of CEA.
CEA is a member of an antigen family. Because of this, the immunoassay of CEA by presently available methods is complicated by the fact that CEA is but one of several potentially reactive antigens. There have been at least sixteen CEA-like antigens described in the literature. Since some of these appear to be the same antigen described by different investigators, the actual number of different antigens is somewhat less than this number. Nonetheless, there is a complex array of cross-reactive antigens which can potentially interfere with an immunoassay of the CEA released by tumors. It is known that serum levels of CEA-like antigens are elevated in many non-cancerous conditions such an inflammatory liver diseases and also in smokers. It is important that immunoassays used for the monitoring of cancer patient status not be interfered with by these other CEA-like antigens. Conversely, it is important to be able to distinguish the antigens by immunoassays because of the possibility that different tumor types may preferentially express different forms of CEA. If so, then the ability to reliably measure the different forms of CEA can provide the means to diagnose or more successfully treat different forms of cancer.
The members of the “CEA family” share some antigenic determinants. These common epitopes are not useful in distinguishing the members of the antigen family and antibodies recognizing them are of little use for measuring tumor-specific CEA levels.
U.S. Pat. No. 3,663,684, entitled “Carcinoembryonic Antigen and Diagnostic Method Using Radioactive Iodine”, concerns purification and radioiodination of CEA for use in a RIA.
U.S. Pat. No. 3,697,638 describes that CEA is a mixture of antigens (components A and B in this case). U.S. Pat. No. 3,697,638 mentions methods for separating and radioiodinating each component and their use in specific RIA's.
U.S. Pat. No. 3,852,415, entitled “Compositions for Use in Radioimmunoassay, as Substitute for Blood Plasma Extract in Determination of Carcinoembryonic Antigen” relates to the use of a buffer containing EDTA and bovine serum albumin as a substitute for plasma as a diluent for CEA RIA's.
U.S. Pat. No. 3,867,363, entitled “Carcinoembryonic Antigens”, is directed to the isolation of CEA components A and B, their labelling and use in a RIA.
U.S. Pat. No. 3,927,193, entitled “Localization of Tumors by Radiolabelled Antibodies”, concerns the use of radiolabelled anti-CEA antibodies in whole body tumor imaging.
U.S. Pat. No. 3,956,258, entitled “Carcinoembryonic Antigens”, relates to the isolation of CEA components A and B.
U.S. Pat. No. 4,086,217, entitled “Carcinoembryonic Antigens”, is directed to the isolation of CEA components A and B.
U.S. Pat. No.4,140,753, entitled “Diagnostic Method and Reagent”, concerns the purification of a CEA isomer called CEA-S1 and its use in a RIA.
U.S. Pat. No. 4,145,336, entitled “Carcinoembryonic Antigen Isomer”, relates to the antigen CEA-S1.
U.S. Pat. No. 4,180,499, entitled “Carcinoembryonic Antigens”, describes a process for producing CEA component B.
U.S. Pat. No. 4,228,236, entitled “Process of Producing Carcinoembryonic Antigen”, is directed to the use of the established cell lines LS-174T and LS-180 or clones or derivatives thereof for the production of CEA.
U.S. Pat. No. 4,272,504, entitled “Antibody Adsorbed Support Method for Carcinoembryonic Antigen Assay”, concerns two concepts for the radioimmunoassay of CEA. First, U.S. Pat. No. 4,272,504 relates to a sample pretreatment in the form of heating to 65 to 85° C. at pH 5 to precipitate and eliminate extraneous protein. Second, it describes the use of a solid phase antibody (either on beads or tubes) as a means to capture analyte and radiolabelled CEA tracer.
U.S. Pat. No. 4,299,815, entitled “Carcinoembryonic Antigen Determination”, concerns diluting a CEA sample with water and pretreating by heating to a temperature below which precipitation of protein will occur. The pretreated sample is then immunoassayed using RIA, EIA, FIA or chemiluminescent immunoassay.
U.S. Pat. No. 4,349,528, entitled “Monoclonal Hybridoma Antibody Specific for High Molecular Weight Carcinoembryonic Antigen”, is directed to a monoclonal antibody reacting with 180 kD CEA, but not with other molecular weight forms.
U.S. Pat. No. 4,467,031, entitled “Enzyme-Immunoassay for Carcinoembryonic Antigen”, relates to a sandwich enzyme immunoassay for CEA in which the first of two anti-CEA monoclonal antibodies is attached to a solid phase and the second monoclonal is conjugated with peroxidase.
U.S. Pat. No. 4,489,167, entitled “Methods and Compositions for Cancer Detection”, describes that CEA shares an antigenic determinant with alpha-acid glycoprotein (AG), which is a normal component of human serum. The method described therein concerns a solid-phase sandwich enzyme immunoassay using as one antibody an antibody recognizing AG and another antibody recognizing CEA, but not AG.
U.S. Pat. No. 4,578,349, entitled “Immunoassay for Carcinoembryonic Antigen (CEA)”, is directed to the use of high salt containing buffers as diluents in CEA immunoassays.
EP 113072-A, entitled “Assaying Blood Sample for Carcinoembryonic Antigen—After Removal of Interfering Materials by Incubation with Silica Gel”, relates to the removal from a serum of a plasma sample of interfering substances by pretreatment with silica gel. The precleared sample is then subjected to an immunoassay.
EP 102008-A, entitled “Cancer Diagnostics Carcinoembryonic Antigen—Produced from Perchloric Acid Extracts Without Electrophoresis”, relates to a procedure for the preparation of CEA from perchloric acid extracts, without the use of an electrophoresis step.
EP 92223-A, entitled “Determination of Carcinoembryonic Antigen in Cytosol or Tissue—for Therapy Control and Early Recognition of Regression”, concerns an immunoassay of CEA, not in serum or plasma, but in the cytosol fraction of the tumor tissue itself.
EP 83103759.6, entitled “Cytosole-CEA-Measurement as Predictive Test in Carcinoma, Particularly Mammacarcinoma”, is similar to EP 92223-A.
EP 83303759, entitled “Monoclonal Antibodies Specific to Carcinoembryonic Antigen”, relates to the production of “CEA specific” monoclonal antibodies and their use in immunoassays.
WO 84/02983, entitled “Specific CEA-Family Antigens, Antibodies Specific Thereto and Their Methods of Use”, is directed to the use of monoclonal antibodies to CEA-meconium (MA)-, and NCA-specific epitopes in immunoassays designed to selectively measure each of these individual components in a sample.
All of the heretofore CEA assays utilize either monoclonal or polyclonal antibodies which are generated by immunizing animals with the intact antigen of choice. None of them address the idea of making sequence specific antibodies for the detection of a unique primary sequence of the various antigens. They do not cover the use of any primary amino acid sequence for the production of antibodies to synthetic peptides or fragments of the natural product. They do not include the concept of using primary amino acid sequences to distinguish the CEA family members. None of them covers the use of DNA or RNA clones for isolating the genes with which to determine the primary sequence.
DEFINITIONS
Nucleic Acid Abbreviations
A adenine
G guanine
C cytosine
T thymidine
U uracil
Amino Acid Abbreviations:
Asp aspartic acid
Asn asparagine
Thr threonine
Ser serine
Glu glutamic acid
Gln glutamine
Pro proline
Gly glycine
Ala alanine
Cys cysteine
Val valine
Met methionine
Ile isoleucine
Leu leucine
Tyr tyrosine
Phe phenylalanine
Trp tryptophan
Lys lysine
His histidine
Arg arginine
Nucleotide—A monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of the pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide. The four DNA bases are adenine (“A”), guanine (“G”), cytosine (“C”), and thymine (“T”). The four RNA bases are A, G, C and uracil (“U”).
DNA Sequence—A linear array of nucleotides connected one to the other by phosphodiester bonds between the 3′ and 5′ carbons of adjacent pentoses.
Functional equivalents—It is well known in the art that in a DNA sequence some nucleotides can be replaced without having an influence on the sequence of the expression product. With respect to the peptide this term means that one or more amino acids which have no function in a particular use can be deleted or replaced by another one.
Codon—A DNA sequence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a translation start signal or a translation termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode the amino acid leucine (“Leu”), TAG, TAA and TGA are translation stop signals and ATG is a translation start signal.
Reading Frame—The grouping of codons during translation of mRNA into amino acid sequences. During translation, the proper reading frame must be maintained. For example, the sequence GCTGGTTGTAAG may be translated in three reading frames or phases, each of which affords a different amino acid sequence
GCT GGT TGT AAG—Ala-Gly-Cys-Lys
G CTG GTT GTA AG—Leu-Val-Val
GC TGG TTG TAA G—Trp-Leu-(STOP).
Polypeptide—A linear array of amino acids connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids.
Genome—The entire DNA of a cell or a virus. It includes inter alia the structural genes coding for the polypeptides of the cell or virus, as well as its operator, promoter and ribosome binding and interaction sequences, including sequences such as the Shine-Delgarno sequences.
Structural Gene—A DNA sequence which encodes through its template or messenger RNA (“mRNA”) a sequence of amino acids characteristics of a specific polypeptide.
Transcription—The process of producing mRNA from a structural gene.
Translation—The process of producing a polypeptide from mRNA.
Expression—The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
Plasmid—A non-chromosomal double-stranded DNA sequence comprising an intact “replicon” such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (TetR) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a “transformant”.
Phage or Bacteriophage—Bacterial virus, many of which consist of DNA sequences encapsulated in a protein envelope or coat (“capsid protein”).
Cloning Vehicle—A plasmid, phage DNA or other DNA sequence which is capable of replicating in a host cell, which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g., replication, production of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance. A cloning vehicle is often called a vector.
Cloning—The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.
Recombinant DNA Molecule or Hybrid DNA—A molecule consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and have the capacity to infect some host cell and be maintained therein.
cDNA Expression Vector—A procaroytic cloning vehicle which also contains sequences of nucleotides that facilitate expression of cDNA sequences in eucaroytic cells. These nucleotides include sequences that function as eucaryotic promoter, alternative splice sites and polyadenylation signals.
Transformation/Transfection—DNA or RNA is introduced into cells in such a way as to allow gene expression. “Infected” referred to herein concerns the introduction of RNA or DNA by a viral vector into the host.
“Injected” referred to herein concerns the microinjection (use of a small syringe) of DNA into a cell.
CEA antigen family (CEA gene family)—a set of genes (gene family) and their products (antigen family) that share nucleotide sequences homologous to partial cDNA LV-7 (CEA -(a)) and as a result of theses similarities also share a subset of their antigenic epitopes. Examples of the CEA antigen family include CEA (=CEA-(b)), transmembrane CEA (TMCEA)=(CEA-(c) and normal crossrecting antigen NCA (=CEA-(d)).
SUMMARY OF THE INVENTION
The present invention concerns the following DNA sequences designed as TM-2 (CEA-(e)), TM-3 (CEA-(f)), TM-4 (CEA-(g)), KGCEA1 and KGCEA2, which code for CEA antigen family peptide sequences or nucleic acids having a base sequence (DNA or RNA) that are hybridizable therewith:
SEQUENCE AND TRANSLATION OF CDNA OF TM-2
        10                  30                  50
         .         .         .         .         .         .
CAGCCGTGCTCGAAGCGTTCCTGGAGCCCAAGCTCTCCTCCACAGGTGAAGACAGGGCCA
        70                  90                 110
         .         .         .         .         .         .
GCAGGAGACACCATGGGGCACCTCTCAGCCCCACTTCACAGAGTGCGTGTACCCTGGCAG
            MetGlyHiSLeuSerAlaProLeuHisArgValArgValProTrpGln
       130                 150                 170
         .         .         .         .         .         .
GGGCTTCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCCAGCTC
GlyLeuLeuLeuThrAlaSerLeuLeuThrPheTrpAsnProProThrThrAlaGlnLeu
       190                 210                 230
         .         .         .         .         .         .
ACTACTGAATCCATGCCATTCAATGTTGCAGAGGGGAAGGAGGTTCTrCTCCTTGTCCAC
ThrThrGluSerMetProPheAsnValAlaGlyGlyLysGluValLeuLeuLeuValHis
       250                 270                 290
         .         .         .         .         .         .
AATCTGCCCCAGCAACTTTTTGGCTACAGCTGGTACAAAGGGGAAAGAGTGGATGGCAAC
AsnLeuProGlnGlyLeuPheGlyTyrSerTrpTysLysGlyGluArgValAspGlyAsn
       310                 330                 350
         .         .         .         .         .         .
CGTCAAATTGTAGGATATGCAATAGGAACTCAACAAGCTACCCCAGGGCCCGCAAACAGC
ArgGlnIleValGlyTyrAlaIleGlyThrGlnGlnAlaThrProGlyProAlaAsnSer
       370                 390                 410
         .         .         .         .         .         .
GGTCGAGAGACAATATACCCCAATGCATCCCTGCTGATCCAGAACGTCACCCAGAATGAC
GlyArgGluThrIleTyrProAsnAlaSerLeuLeuIleGlnAsnValThrGlnAsnAsp
       430                 450                 470
         .         .         .         .         .         .
ACAGGATTCTACACCCTACAAGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGA
ThrGlyPheTyrThrLeuGlnValIleLysSerAspLeuValAsnGluGluAlaThrGly
       490                 510                 530
         .         .         .         .         .         .
CAGTTCCATGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAACCCT
GlnPheHisValTyrProGluLeuProLysProSerIleSerSerAsnAsnSerAsnPro
       550                 570                 590
         .         .         .         .         .         .
GTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACACAACCTAC
ValGluAspLysAspAlaValAlaPheThrCysGluProGluThrGlnAspThrThrTyr
       610                 630                 650
         .         .         .         .         .         .
CTGTGGTGGATAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGC
LeuTrpTrpIleAsnAsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnGly
       670                 690                 710
         .         .         .         .         .         .
AACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGACACAGGACCCTATGAGTGTGAA
AsnArgThrLeuThrLeuLeuSerValThrArgAsnAspThrGlyProTyrGluCysGlu
       730                 750                 770
         .         .         .         .         .         .
ATACAGAACCCAGTGAGTGCGAACCGCAGTGACCCAGTCACCTTGAATGTCACCTATGGC
IleGlnAsnProValSerAlaAsnArgSerAspProValThrLeuAsnValThrTyrGly
       790                 810                 830
         .         .         .         .         .         .
CCGGACACCCCCACCATTTCCCCTTCAGACACCTATTACCGTCCAGGGGCAAACCTCAGC
ProAspThrProThrIleSerProSerAspThrTyrTyrArgProGlyAlaAsnLeuSer
       850                 870                 890
         .         .         .         .         .         .
CTCTCCTGCTATGCAGCCTCTAACCCACCTGCACAGTACTCCTGGCTTATCAATGGAACA
LeuSerCysTyrAlaAlaSerAsnProProAlaGlnTyrSerTrpLeuIleAsnGlyThr
       910                 930                 950
         .         .         .         .         .         .
TTCCAGCAAAGCACACAAGAGCTCTTTATCCCTAACATCACTGTGAATAATAGTGGATCC
PheGlnGlnSerThrGlnGluLeuPheIleProAsnIleThrValAsnAsnSerGlySer
       970                 990                1010
         .         .         .         .         .         .
TATACCTGGACGCCAATAACTCAGTCACTTGGCTGCAACAGGACCACAGTCAAGACGATC
TyrThrCysHisAlaAsnAsnSerValThrGlyCysAsnArgThrThrValLysThrIle
      1030                1050                1070
         .         .         .         .         .         .
ATAGTCACTGATAATGCTCTACCACAAGAAAATGGCCTCTCACCTGGGGCCATTGCTGGC
IleValThrAspAsnAlaLeuProGlnGluAsnGlyLeuSerProGlyAlaIleAlaGly
      1090                1110                1130
         .         .         .         .         .         .
ATTGTGATTGGAGTAGTGGCCCTGGTTGCTCTGATAGCAGTAGCCCTGGCATGTTTTCTG
IleValIleGlyValValAlaLeuValAlaLeuIleAlaValAlaLeuAlaCysPheLeu
      1150                1170                1190
         .         .         .         .         .         .
CATTTCGGGAAGACCGGCAGGGCAAGCGACCAGCGTGATCTCACAGAGCACAAACCCTCA
HisPheGlyLysThrGlyArgAlaSerAspGlnArgAspLeuThrGluHisLysProSer
      1210                1230                1250
         .         .         .         .         .         .
GTCTCCAACCACACTCAGGACCACTCCAATGACCCACCTAACAAGATGAATGAAGTTACT
ValSerAsnHisThrGlnAspHisSerAsnAspProProAsnLysMetAsnGluValThr
      1270                1290                1310
         .         .         .         .         .         .
TATTCTACCCTGAACTTTGAAGCCCAGCAACCCACACAACCAACTTCAGCCTCCCCATCC
TyrSerThrLeuAsnPheGluAlaGlnGlnProThrGlnProThrSerAlaSerProSer
      1330                1350                1370
         .         .         .         .         .         .
CTAACAGCCACAGAAATAATTTATTCAGAAGTAAAAAAGCAGTAATGAAACCTGTCCTGC
LeuThrAlaThrGluIleIleTyrSerGluValLysLysGln
      1390                1410                1430
         .         .         .         .         .         .
TCACTGCAGTGCTGATGTATTTCAAGTCTCTCACCCTCATCACTAGGAGATTCCTTTCCC
      1450                1470                1490
         .         .         .         .         .         .
CTGTAGGGTAGAGGGGTGGGGACAGAAACAACTTTCTCCTACTCTTCCTTCCTAATAGGC
      1510                1530                1550
         .         .         .         .         .         .
ATCTCCAGGCTGCCTGGTCACTGCCCCTCTCTCAGTGTCAATAGATGAAAGTACATTGGG
      1570                1590                1610
         .         .         .         .         .         .
AGTCTGTAGGAAACCCAACCTTCTTGTCATTGAAATTTGGCAAAGCTGACTTTGGGAAAG
      1630                1650                1670
         .         .         .         .         .         .
AGGGACCAGAACTTCCCCTCCCTTCCCCTTTTCCCAACCTGGACTTGTTTTAAACTTGCC
      1690                1710                1730
         .         .         .         .         .         .
TGTTCAGAGCACTCATTCCTTCCCACCCCCAGTCCTGTCCTATCACTCTAATTCGGATTT
      1750                1770                1790
         .         .         .         .         .         .
GCCATAGCCTTGAGGTTATGTCCTTTTCCATTAAGTACATGTGCCAGGAAACAGCGAGAG
      1810                1830                1850
         .         .         .         .         .         .
AGAGAAAGTAAACGGCAGTAATGCTTCTCCTATTTCTCCAAAGCCTTGTGTGAACTAGCA
      1870                1890                1910
         .         .         .         .         .         .
AAGAGAAGAAAATCAAATATATAACCAATAGTGAAATGCCACAGGTTTGTCCACTGTCAG
      1930                1950                1970
         .         .         .         .         .         .
GGTTGTCTACCTGTAGGATCAGGGTCTAAGCACCTTGGTGCTTAGCTAGAATACCACCTA
      1990                2010                2030
         .         .         .         .         .         .
ATCCTTCTGGCAAGCCTGTCTTCAGAGAACCCACTAGAAGCAACTAGGAAAAATCACTTG
      2050                2070                2090
         .         .         .         .         .         .
CCAAAATCCAAGGCAATTCCTGATGGAAAATGCAAAAGCACATATATGTTTTAATATCTT
      2110                2130                2150
         .         .         .         .         .         .
TATGGGCTCTGTTCAAGGCAGTGACTGAGAGGGAGGGGTTATAGCTCAGGAGGGAACCAG
      2170                2190                2210
         .         .         .         .         .         .
CTTCTGATAAACACAATCTGCTAGGAACTTGGGAAAGGAATCAGAGAGCTGCCCTTCAGC
      2230                2250                2270
         .         .         .         .         .         .
GATTATTTAAATTGTTAAAGAATACACAATTTGGGGTATTGGGATTTTTCTCCTTTTCTC
      2290                2310                2330
         .         .         .         .         .         .
TGAGACATTCCACCATTTTAATTTTTGTAACTGCTTATTTATGTGIAAAGGGTTATTTTT
      2350                2370                2390
         .         .         .         .         .         .
ACTTAGCTTAGCTATGTCAGCCAATCCGATTGCCTTAGGTGAAAGAAACCACCGAAATCC
      2410                2430                2450
         .         .         .         .         .         .
CTCAGGTCCCTTGGTCAGGAGCCTCTCAAGATTTTTTTTGTCAGAGGCTCCAAATAGAAA
      2470                2490                2510
         .         .         .         .         .         .
ATAAGAAAAGGTTTTCTTCATTCATGGCTAGAGCTAGATTTAACTCAGTTTCTAGGCACC
      2530                2550                2570
         .         .         .         .         .         .
TCAGACCAATCATCAACTACCATTCTATTCCATGTTTGCACCTGTGCATTTTCTGTTTGC
      2590                2610                2630
         .         .         .         .         .         .
CCCCATTCACTTTGTCAGGAAACCTTGGCCTCTGCTAAGGTGTATTTGGTCCTTGAGAAG
      2650                2670                2690
         .         .         .         .         .         .
TGGGAGCACCCTACAGGGACACTATCACTCATGCTGGTGGCATTGTTTACAGCTAGAAAG
      2710                2730                2750
         .         .         .         .         .         .
CTGCACTGGTGCTAATGCCCCTTGGGAAATGGGGCTGTGAGGAGGAGGATTATAACTTAG
      2770                2790                2810
         .         .         .         .         .         .
GCCTAGCCTCTTTTAACAGCCTCTGAAATTTATCTTTTCTTCTATGGGGTCTATAAATGT
      2830                2850                2870
         .         .         .         .         .         .
ATCTTATAATAAAAAGGAAGGACAGGAGGAAGACAGGCAAATGTACTTCTCACCCAGTCT
      2890                2910                2930
         .         .         .         .         .         .
TCTACACAGATGGAATCTCTTTGGGGCTAAGAGAAAGGTTTTATTCTATATTGCTTACCT
      2950                2970                2990
         .         .         .         .         .         .
GATCTCATGTTAGGCCTAAGAGGCTTTCTCCAGGAGGATTAGCTTGGAGTTCTCTATACT
      3010                3030                3050
         .         .         .         .         .         .
CAGGTACCTCTTTCAGGGTTTTCTAACCCTGACACGGACTGTGCATACTTTCCCTCATCC
      3070                3090                3110
         .         .         .         .         .         .
ATGCTGTGCTGTGTTATTTAATTTTTCCTGGCTAAGATCATGTCTGAATTATGTATGAAA
      3130                3150                3170
         .         .         .         .         .
ATTATTCTATGTTTTTATAATAAAAATAATATATCAGACATCGAAAAAAAAAA
SEQUENCE AND TRANSLATION OF cDNA OF TM-3
        10                  30                  50
         .         .         .         .         .         .
CAGCCGTGCTCGAAGCGTTCCTGGAGCCCAAGCTCTCCTCCACAGGTGAAGACAGGGCCA
        70                  90                 110
         .         .         .         .         .         .
GCAGGAGACACCATGGGGCACCTCTCAGCCCCACTTCACAGAGTGCGTGTACCCTGGCAG
            MetGlyHisLeuSerAlaProLeuHisArgValArgValProTrpGln
       130                 150                 170
         .         .         .         .         .         .
GGGCTTCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCCAGCTC
GlyLeuLeuLeuThrAlaSerLeuLeuThrPheTrpAsnProProThrThrAlaGlnLeu
       190                 210                 230
         .         .         .         .         .         .
ACTACTGAATCCATGCCATTCAATGTTGCAGAGGGGAAGGAGGTTCTTCTCCTTGTCCAC
ThrThrGluSerIleProPheAsnValAlaGluGlyLysGluValLeuLeuLeuValHis
       250                 270                 290
         .         .         .         .         .         .
AATCTGCCCCAGCAACTTTTTGGCTACAGCTGGTACAAAGGGGAAAGAGTGGATGGCAAC
AsnLeuProGlnGlnLeuPheGlyTyrSerTrpTyrLysGlyGluArgValAspGlyAsn
       310                 330                 350
         .         .         .         .         .         .
CGTCAAATTGTAGGATATGCAATAGGAACTCAACAAGCTACCCCAGGGCCCGCAAACAGC
ArgGlnIleValGlyTyrAlaIleGlyThrGlnGlnAlaThrProGlyProAlaAsnSer
       370                 390                 410
         .         .         .         .         .         .
GGTCGAGAGACAATATACCCCAATGCATCCCTGCTGATCCAGAACGTCACCCAGAATGAC
GlyArgGluThrIleTyrProAsnAlaSerLeuLeuIleGlnAsnValThrGlnAsnAsp
       430                 450                 470
         .         .         .         .         .         .
ACAGGATTCTACACCCTACAAGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGA
ThrGlyPheTyrThrLeuGlnValIleLysSerAspLeuValAsnGluGluAlaThrGly
       490                 510                 530
         .         .         .         .         .         .
CAGTTCCATGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAACCCT
GlnPheHisValTyrProGluLeuProLysProSerIleSerSerAsnAsnSerAsnPro
       550                 570                 590
         .         .         .         .         .         .
GTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACACAACCTAC
ValGluAspLysAspAlaValAlaPheThrCysGluProGluThrGlnAspThrThrTyr
       610                 630                 650
         .         .         .         .         .         .
CTGTGGTGGATAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGC
LeuTrpTrpIleAsnAsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnGly
       670                 690                 710
         .         .         .         .         .         .
AACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGACACAGGACCCTATGAGTGTGAA
AsnArgThrLeuThrLeuLeuSerValThrArgAsnAspThrGlyProTyrGluCysGlu
       730                 750                 770
         .         .         .         .         .         .
ATACAGAACCCAGTGAGTGCGAACCGCAGTGACCCAGTCACCTTGAATGTCACCTATGGC
IleGlnAsnProValSerAlaAsnArgSerAspProValThrLeuAsnValThrTyrGly
       790                 810                 830
         .         .         .         .         .         .
CCGGACACCCCCACCATTTCCCCTTCAGACACCTATTACCGTCCAGGGGCAAACCTCAGC
ProAspThrProThrIleSerProSerAspThrTyrTyrArgProGlyAlaAsnLeuSer
       850                 870                 890
         .         .         .         .         .         .
CTCTCCTGCTATGCAGCCTCTAACCCACCTGCACAGTACTCCTGGCTTATCAATGGAACA
LeuSerCysTyrAlaAlaSerAsnProProAlaGlnTyrSerTrpLeuIleAsnGlyThr
       910                 930                 950
         .         .         .         .         .         .
TTCCAGCAAAGCACACAAGAGCTCTTTATCCCTAACATCACTGTGAATAATAGTGGATCC
PheGlnGlnSerThrGlnGlnLeuPheIleProAsnIleThrValAsnAsnSerGlySer
       970                 990                1010
         .         .         .         .         .         .
TATACCTGCCACGCCAATAACTCAGTCACTGGCTGCAACAGGACCACAGTCAAGACGATC
TyrThrCysHisAlaAsnAsnSerValThrGlyCysAsnArgThrThrValLysThrIle
      1030                1050                1070
         .         .         .         .         .         .
ATAGTCACTGAGCTAAGTCCAGTAGTAGCAAAGCCCCAAATCAAAGCCAGCAAGACCACA
IleValThrGluLeuSerProValAlaLysProGlnIleLysLysAlaSerLysThrThr
      1090                1110                1130
         .         .         .         .         .         .
GTCACAGGAGATAAGGACTCTGTGAACCTGACCTGCTCCACAAATGACACTGGAATCTCC
ValThrGlyAspLysAspSerValAsnLeuThrCysSerThrAsnAspThrGlyIleSer
      1150                1170                1190
         .         .         .         .         .         .
ATCCGTTGGTTCTTCAAAAACCAGAGTCTCCCGTCCTCGGAGAGGATGAAGCTGTCCCAG
IleArgTrpPhePheLysAsnGlnSerLeuProSerSerGluArgMetLysLeuSerGln
      1210                1230                1250
         .         .         .         .         .         .
GGCAACACCACCCTCAGCATAAACCCTGTCAAGAGGGAGGATGCTGGGACGTATTGGTGT
GlyAsnThrThrLeuSerIleAsnProValLysArgGluAspAlaMetLysLeuSerGln
      1270                1290                1310
         .         .         .         .         .         .
GAGGTCTTCAACCCAATCAGTAAGAACCAAAGCGACCCCATCATGCTGAACGTAAACTAT
GluValPheAsnProIleSerLysAsnGlnSerAspProIleMetLeuAsnValAsnTyr
      1330                1350                1370
         .         .         .         .         .         .
AATGCTCTACCACAAGAAAATGGCCTCTCACCTGGGGCCATTGCTGGCATTGTGATTGGA
AsnAlaLeuProGlnGlnAsnGlyLeuSerProGlyAlaIleAlaGlyIleValIleGly
      1390                1410                1430
         .         .         .         .         .         .
GTAGTGGCCCTGGTTGCTCTGATAGCAGTAGCCCTGGCATGTTTTCTGCATTTCGGGAAG
ValValAlaLeuValAlaLeuIleAlaValAlaLeuAlaCysPheLeuHisPheGlyLys
      1450                1470                1490
         .         .         .         .         .         .
ACCGGCAGCTCAGGACCACTCCAATGACCCACCTAACAAGATGAATGAAGTTACTTATTC
ThrGlySerSerGlyProLeuGln
      1510                1530                1550
         .         .         .         .         .         .
TACCCTGAACTTTGAAGCCCAGCAACCCACACAACCAACTTCAGCCTCCCCATCCCTAAC
      1570                1590                1610
         .         .         .         .         .         .
AGCCACAGAAATAATTTATTCAGAAGTAAAAAAGCAGTAATGAAACCTGAAAAAAAAAAA
      1630
         .
AAAAAAAAAA
SEQUENCE AND TRANSLATION OF CDNA OF TM-4
        10                  30                  50
         .         .         .         .         .         .
CAGCCGTGCTCGAAGCGTTCCTGGAGCCCAAGCTCTCCTCCACAGGTGAAGACAGGGCCA
        70                  90                 110
         .         .         .         .         .         .
GCAGGAGACACCATGGGGCACCTCTCAGCCCCACTTCACAGAGTGCGTGTACCCTGGCAG
            MetGlyHisLeuSerAlaProLeuHisArgValArgValProTrpGln
       130                 150                 170
         .         .         .         .         .         .
GGGCTTCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCCAGCTC
GlyLeuLeuLeuThrAlaSerLeuLeuThrPheTrpAsnProProThrThrAlaGlnLeu
       190                 210                 230
         .         .         .         .         .         .
ACTACTGAATCCATGCCATTCAATGTTGCAGAGGGGAAGGAGGTTCTTCTCCTTGTCCAC
ThrThrGluSerMetProPheAsnValAlaGluGlyLysGlyValLeuLeuLeuValHis
       250                 270                 290
         .         .         .         .         .         .
AATCTGCCCCAGCAACTTTTTGGCTACAGCTGGTACAAGGGGIkAAGAGTGGATGGCAAC
AsnLeuProGlnGlnLeuPheGlyTyrSerTrpTyrLysGlyGlyArgValAspGlyAsn
       310                 330                 350
         .         .         .         .         .         .
CGTCAAATTGTAGGATATGCAATAGGAACTCAACAAGCTACCCCAGGGCCCGCAAACAGC
ArgGlnIleValGlyTyrAlaIleGlyThrGlnGlnAlaThrProGlyProAlaAsnSer
       370                 390                 410
         .         .         .         .         .         .
GGTCGAGAGACAATATACCCCAATGCATCCCTGCTGATCCAGAACGTCACCCAGAATGAC
GlyArgGluThrIleTyrProAsnAlaSerLeuLeuIleGlnAsnValThrGlnAsnAsp
       430                 450                 470
         .         .         .         .         .         .
ACAGGATTCTACACCCTACAAGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGA
ThrGlyPheTyrThrLeuGlnValIleLysSerASpLeuValAsnGluGluAlaThrGly
       490                 510                 530
         .         .         .         .         .         .
CAGTTCCATGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAACCCT
GlnPheHisValTyrProGluLeuProLysProSerIleSerSerAsnAsnSerAsnPro
       550                 570                 590
         .         .         .         .         .         .
GTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACACAACCTAC
ValGluAspLysAspAlaValAlaPheThrCysGluProGluThrGlnAspThrThrTyr
       610                 630                 650
         .         .         .         .         .         .
CTGTGGTGGATAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGC
LeuTrpTrpIleAsnAsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnGly
       670                 690                 710
         .         .         .         .         .         .
AACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGACACAGGACCCTATGAGTGTGAA
AsnArgThrLeuThrLeuLeuSerValThrArgAsnAspThrGlyProTyrGluCysGlu
       730                 750                 770
         .         .         .         .         .         .
ATACAGAACCCAGTGAGTGCGAACCGCAGTGACCCAGTCACCTTGAATGTCACCTATGGC
IleGlnAsnProValSerAlaAsnArgSerAspProValThrLeuAsnValThrTyrGly
       790                 810                 830
         .         .         .         .         .         .
CCGGACACCCCCACCATTTCCCCTTCAGACACCTATTACCGTCCAGGGGCAAACCTCAGC
ProAspThrProThrIleSerProSerAspThrTyrTyrArgProGlyAlaAsnLeuSer
       850                 870                 890
         .         .         .         .         .         .
CTCTCCTGCTATGCAGCCTCTAACCCACCTGCACAGTACTCCTGGCTTATCAATGGAACA
LeuSerCysTyrAlaAlaSerAsnProProAlaGlnTyrSerTrpLeuIleAsnGlyThr
       910                 930                 950
         .         .         .         .         .         .
TTCCAGCAAAGCACACAAGAGCTCTTTATCCCTAACATCACTGTGAATAATAGTGGATCC
PheGlnGlnSerThrGlnGluLeuPheIleProAspIleThrValAsnAsnSerGlySer
       970                 990                1010
         .         .         .         .         .         .
TATACCTGCCACGCCAATAACTCAGTCACTGGCTGCAACAGGACCACAGTCAAGACGATC
TyrThrCysHisAlaAsnAsnSerValThrGlyCysAsnArgThrThrValLysThrIle
      1030                1050                1070
         .         .         .         .         .         .
ATAGTCACTGATAATGCTCTACCACAAGAAAATGGCCTCTCACCTGGGGCCATTGCTGGC
IleValThrAspAsnAlaLeuProGlnGlnAsnGlyLeuSerProGlyAlaIleAlaGly
      1090                1110                1130
         .         .         .         .         .         .
ATTGTGATTGGAGTAGTGGCCCTGGTTGCTCTGATAGCAGTAGCCCTGGCATGTTTTCTG
IleValIleGlyValValAlaLeuValAlaLeuIleAlaValAlaLeuAlaCysPheLeu
      1150                1170                1190
         .         .         .         .         .         .
CATTTCGGGAAGACCGGCAGCTCAGGACCACTCCAATGACCCACCTAACAAGATGAATGA
HisPheGlyLysThrGlySerSerGlyProLeuGln
      1210                1230                1250
         .         .         .         .         .         .
AGTTACTTATTCTACCCTGAACTTTGAAGCCCAGCAACCCACACAACCAACTTCAGCCTC
      1270                1290                1310
         .         .         .         .         .         .
CCCATCCCTAACAGCCACAGAAATAATTTATTCAGAAGTAAAAAAGCAGTAATGAAACCT
      1330
         .
GAAAAAAAAAAAAAAAAAA
The present invention is also directed to a replicable recombinant cloning vehicle (“vector”) having an insert comprising a nucleic acid, e.g., DNA, which comprises a base sequence which codes for a CEA peptide or a base sequence hybridizable therewith.
This invention also relates to a cell that is transformed/transfected, infected or injected with the above described replicable recombinant cloning vehicle or nucleic acid hybridizable with the aforementioned cDNA. Thus the invention also concerns the transfection of cells using free nucleic acid, without the use of a cloning vehicle.
Still further, the present invention concerns a polypeptide expressed by the above described transfected, infected or injected cell, which polypeptide exhibits immunological cross-reactivity with a CEA, as well as labelled forms of the polypeptide. The invention also relates to polypeptides having an amino acid sequence, i.e., synthetic peptides, or the expression product of a cell that is transfected, injected, infected with the above described replicable recombinant cloning vehicles, as well as labelled forms thereof. Stated otherwise, the present invention concerns a synthetic peptide having an amino acid sequence corresponding to the entire amino acid sequence or a portion thereof having no less than five amino acids of the aforesaid expression product.
The invention further relates to an antibody preparation specific for the above described polypeptide.
Another aspect of the invention concerns an immunoassay method for detecting CEA or a functional equivalent thereof in a test sample comprising
(a) contacting the sample with the above described antibody preparation, and
(b) determining binding thereof to CEA in the sample.
The invention also is directed to a nucleic acid hybridization method for detecting a CEA or a related nucleic acid (DNA or RNA) sample in a test sample comprising
(a) contacting the test sample with a nucleic acid probe comprising a nucleic acid, which comprises a base sequence which codes for a CEA peptide sequence or a base sequence that is hybridizable therewith, and
(b) determining the formation of the resultant hybridized probe.
The present invention also concerns a method for detecting the presence of carcinoembryonic antigen or a functional equivalent thereof in an animal or human patient in vivo comprising
a) introducing into said patient a labeled (e.g., a radio-opaque material that can be detected by X-rays, radiolabeled or labeled with paramagnetic materials that can be detected by NMR) antibody preparation according to the present invention and
b) detecting the presence of such antibody preparation in the patient by detecting the label.
In another aspect, the present invention relates to the use of an antibody preparation according to the present invention for therapeutic purposes, namely, attaching to an antibody preparation radionuclides, toxins or other biological effectors to form a complex and introducing an effective amount of such complex into an animal or human patient, e.g., by injection or orally. The antibody complex would attach to CEA in a patient and the radionuclide, toxin or other biological effector would serve to destroy the CEA expressing cell.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of the transmembrane CEA's.
DETAILED DESCRIPTION OF THE INVENTION
In patent applications, applicants described the following CEA's:
ATCC No.
CEA-(a) partial CEA (pcLV7)
CEA-(b) full coding CEA (pc 15LV7) 67709
CEA-(c) TM-1 (FL-CEA; pc 19-22) 67710
CEA-(d) NCA (pcBT 20) 67711
In the present application, applicants described the following CEA's:
ATCC No.
CEA-(e) TM-2 (pc E22) 67712
CEA-(f) TM-3 (pc HT-6) 67708
CEA-(g) TM-4.
ATCC Nos. 67708, 67709, 67710, 67711 and 67712 were all deposited with the American Type Culture Collection on May 25, 1988.
The sequences for CEA-(a), CEA-(b), CEA-(c) and CEA-(d) are given hereinbelow:
CEA-(a):
GG GGT TTA CAC AAC CAC CAC CCC ATC AAA CCC TTC ATC ACC AGC AAC AAC TCC AAC CCC GTG
   GAG GAT GAG GAT GCT GTA GCC TTA ACC TGT GAA CCT GAG ATT CAG AAC ACA ACC TAC CTG
   TGG TGG GTA AAT AAT CAG AGC CTC CCG GTC AGT CCC AGG CTG CAG CTG TCC AAT GAC AAC
   AGG ACC CTC ACT CTA CTC AGT GTC ACA AGG AAT GAT GTA GGA CCC TAT GAG TGT GGA ATC
   CAG AAC GAA TTA AGT GTT GAC CAC AGC GAC CCA GTC ACC CAG CGA TTC CTC TAT GGC CCA
   GAC GAC CCC ACC ATT TCC CCC TCA TAC ACC TAT TAC CGT CCA GGG GTG GAA CCT CAG CCT
   CTC TGC CAT GCA GCC TCT AAC CCA CCT GCA CAG TAT TCT TGG CTG ATT GAT GGG ACC GTC
   CAG CAA CAC ACA CAA GAG CTC TTT ATC TCC AAC ATC ACT GAG AAG AAC AGC GGA CTC TAT
   ACC TGC CAG GCC AAT AAC TCA GCC AGT GGC ACA GCA GGA CTA CAG TCA AGA CAA TCA CAG
   TCT CTG CGG ATG CCC AAG CCC TCC ATC TCC AGC AAC AAC TCC AAA CCC GTG GAG GAC AAG
   GAT CGC TGT GGC CTT CAC TGT GAA CCT GAG GCT CAG AAC ACA ACC TAC CTG TGG TGG GTA
   AAT GGT CAG AGC CTC CCA GTC AGT CCC AGG CTG CAG CTG TCC AAT GGC AAC AGG ACC CTC
   ACT CTA TTC AAT GTC ACA AGA AAT GAC GCA AGA GCC TAT GTA TGT GGA ATC CAG AAC TCA
   GTG AGT GCA AAC CGC AGT GAC CCA GTC ACC CTG GAT GTC CTC TAT GGG CCG GAC ACC CCC
   ATC ATT TCC CCC CCC CC
CEA-(b):
            10            20           30           40            50
            *             *            *            *             *
C ACC ATG GAG TCT CCC TCU GCC CCT CTC CAC ACA TGG TGC ATC CCC TGU CAG AGG CTC
      Met Glu Ser Pre Ser Ala Pro Leu His Arg Typ Cys Ile Pro Trp Gln Arg Leu
   60           70            80           90          100           110
   *            *             *            *            *             *
  CTG CTC ACA OCC TCA CTT CTA ACC TTC TGG AAC CCG CCC ACC ACT GCC AAG CTC ACT
  Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asp Pro Pro Thr Thr Ala Lys Leu Thr
      120          130           140          150          160           170
       *            *             *            *            *             *
  ATT GAA TCC ACG CCG TTC AAT GTC GCA GAG GGC AAG GAG GTO CTT CTA CTT GTC CAC
  Iln Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu Val Leu Leu Leu Val His
          180          190           200          210          220
           *            *             *            *            *
  CAT CTG CCC CAG CAT CTT TTT CCC TAC AGC TGG TAC AAA GGT GAA AGA GTG CAT GGC
  Asn Arg Gln Ile Ile Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala
 230          240          250           260          270          280
  *            *            *             *            *            *
  AAC CGT CAA ATT ATA GGA TAT GTA ATA GGA ACT CAA CAA GCT ACC CCA GGG CCC GCA
  Asn Arg Gln Ile Ile Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala
     290          300          310           320          330          340
      *            *            *             *            *            *
  TAC AGT GGT CGA GAG ATA ATA TAC CCC AAT GCA TCC CTG CTG ATC CAG GAC ATC ATC
  Tyr Ser Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile Ile
         350          360          370           380          390          400
          *            *            *             *            *           *
  GAG AAT GAC ACA GGA TTC TAC ACC CTA CAC GTC ATA AAG TCA GAT CTT GTG AAT CAA
  Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp Leu Val Asn Gly
             410          420          430           440          450
              *            *            *             *            *
  GAA CCA ACA GCC CAG TTC GGG GTA TAC GGG GAG CTG CCC AAG CCC TCC ATC TCC AGC
  Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Gly Leu Pro Lys Pro Ser Ile Ser Ser
   460           470          480          490           500          510
    *             *            *            *             *            *
  AAC AAC TCC AAA CCC GTG GAG GAC AAG CAT GCT GTG CCC TTC ACC TGT CAA CCT GAG
  Asn Asn Ser Lys Pro Val Gly Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu
       520           530          540          550           560          570
        *             *            *            *             *            *
  ACT CAG GAC CCA ACC TAC CTG TGG TGG GTA AAC AAT CAG AAG CTC CCG CTC AGT CCC
  Thr Gln Asp Ala Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro
           580           590          600          610           620
            *             *            *            *             *
  AGG CTG CAG CTG TCC AAT GCC AAC AGG ACC CTC ACT CTA TTC AAT GTC ACA AGA AAT
  Arg Leu Gln Leu Ser Asn Gly Asp Arg Leu Thr Leu Thr Phe Asn Val Thr Arg Asn
  630          640           650          660          670           680
   *            *             *            *            *             *
  GAA CAA GCA AGC TAC AAA TGT CAA ACC CAG AAC CCA GTG AGT GCC AGG CGC AGT GAT
  Glu Gln Ala Ser Tyr Lys Cys Gly Thr Gln Asn Pro Val Ser Ala Arg Arg Ser Asp
      690          700           710          720          730           740
       *            *             *            *            *             *
  TCA GTC ATC CTG AAT GTC CTC TAT GGC CCG GAT GCC CCC ACC ATT TCC CCT CTA AAC
  Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro Thr Ile Ser Pro Leu Asn
          750          760           770          780          790
           *            *             *            *            *
  ACA TCT TAC AGA TCA GGG GAA AAT CTG AAC CTC TCC TGC CAC GCA GCC TCT AAC CCA
  Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn Leu Ser Cys His Ala Ala Ser Asn Pro
 800          810          820           830          840          850
  *            *            *             *            *            *
  CCT GCA CAG TAC TCT TGG TTT GTC AAT GGG ACT TTC CAG CAA TCC ACC CAA GAG CTC
  Pro Ala Gln Tyr Ser Trp Phe Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu
     860          870          880           890          900          910
      *            *            *             *            *            *
  TTT ATC CCC AAC ATC ACT GTE AAT AAT AGT GGA TCC TAT ACG TGC CAA GCC CAT AAC
  Phe Ile Pro Asn Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn
         920          930          940           950          960          970
          *            *            *             *            *            *
  TCA GAC ACT GGC CTC AAT AGG ACC ACA GTC ACG ACG ATC ACA GTC TAT GCA GAG CCA
  Ser Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala Glu Pro
             980          990         1000          1010         1020
              *            *            *             *            *
  CCC AAA CCC TTC ATC ACC AGC AAC AAC TCC AAC CCC GTG GAG GAT GAG GAT GCT GTA
  Ser Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala Glu Pro
  1030          1040         1050         1060          1070         1080
    *             *            *            *             *            *
  GCC TTA ACC TGT GAA CCT GAG ATT CAG AAC ACA ACC TAC CTG TGG TGG GTA AAT AAT
  Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr Thr tyr Leu Trp Trp Val Asn Asn
      1090          1100         1110         1120          1130         1140
        *             *            *            *             *            *
  CAG AGC CTC CCG GTC AGT CCC AGG CTG CAG CTG TCC AAT GAC AAC AGG ACC CTC ACT
  Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser Asn Asp Asn Arp Thr Leu Thr
          1150          1160         1170         1180          1190
            *             *            *            *             *
  CTA CTE AGT GTC ACA AGG AAT GAT ATA GGA CCC TAT GAG TGT GCA ATC CAG AAC GAA
  Leu Leu Ser Val Thr Arg Asn Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asn Asp
 1200         1210          1220         1230         1240          1250
   *            *             *            *            *             *
  TTA AGT GTT GAC CAC AGC GAC CCA GTC ATC CTG AAT GTC CTC TAT GGC CCA GAC GAC
  Leu Ser Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asn Asp
     1260         1270          1280         1290         1300          1310
       *            *             *            *            *             *
  CCC ACC ATT TCC CCC TCA TAC ACC TAT TAC CGT CCA GGG GTG AAC CTC AGC CTC TCC
  Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn Leu Ser Leu Ser
         1320         1330          1340         1350         1360
           *            *             *            *            *
  TGC CAT GCA GCC TCT AAC CCA CCT GCA CAG TAT TCT TGG CTG ATT CAT GGG AAC ATC
  Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu Ile Asp Gly Asn Ile
1370         1380         1390          1400         1410         1420
  *            *            *             *            *            *
  CAG CAA CAC ACA CAA AAG CTC TTT ATC TCC AAC ATC ACT GAG AAC AAC AGC GGA CTC
  Gln Gln His Thr Gln Glu Leu Phe Ile Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu
    1430         1440         1450          1460         1470         1480
      *            *            *             *            *            *
  TAT ACC TGC CAG GCC AAT AAC TCA GCC AGT GGC CAC AGC AGG ACT ACA GTC AAG ACA
  Tyr Thr Cys Gln Ala Asn Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr
        1490         1500         1510          1520         1530         1540
          *            *            *             *            *            *
  ATC ACA GTC TCT GCG GAC GTG CCC GAG CCC TCC ATC TCC AGC AAC AAC TCC AAA CCC
  Ile Thr Val Ser Ala Asp Val Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro
            1550         1560         1570          1580         1590
              *            *            *             *            *
  GTG GAG GAC AAG GAT GCT GTG GCG TTC ACC TGT GAA CCT GAG GCT CAG AAC ACA ACC
  Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln Asn Thr Thr
  1600          1610         1620         1630          1640         1650
    *             *            *            *             *            *
  TAC CTG TGG TGG GTA AAT GGT CAG AGC CTC CCA GTC AGT CCC AGG CTG CAG CTG TCC
  Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser
      1660          1670         1680         1690          1700         1710
        *             *            *            *             *            *
  AAT GGC AAC AGG ACC GTC ACT CTA TTC AAT GTC ACA AGA AAT GAC GCA AGA GCC TAT
  Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr
          1720          1730         1740         1750          1760
            *             *            *            *             *
  GTA TGT GGA ATC CAG AAC TCA GTG AGT GCA AAC CGC AGT GAC CCA GTC ACC CTG GAT
  Val Cys Gly Ile Gln Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp
 1770         1780          1790         1800         1810          1820
   *            *             *            *            *             *
  GTC CTC TAT GGG CCG GAC ACC CCC ATC ATT TCC CCC CCA GAC TCG TCT TAC CTT TCG
  Val Leu Tyr Gly Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser
     1830         1840          1850         1860         1870          1880
       *            *             *            *            *             *
  GGA GCG AAC CTC AAC CTC TCC TGC CAC TCA GCC TCT AAC CCA TCC CCG CAG TAT TCT
  Gly Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln Tyr Ser
         1890         1900          1910         1920         1930
           *            *             *            *            *
  TGG CGT ATC AAT GGG ATA CCG CAG CAA CAC ACA CAA GTT CTC TTT ATC GCC AAA ATC
  Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu Phe Ile Ala Lys Ile
1940         1950         1960          1970         1980         1990
  *            *            *             *            *            *
  ACG CCA AAT AAT AAC GGG ACC TAT GCC TGT TTT GTC TCT AAC TTG GCT ACT GGC CGC
  Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg
    2000         2010         2020          2030         2040         2050
      *            *            *             *            *            *
  AAT AAT TCC ATA GTC AAG AGC ATG ACA GTC TCT GCA TCT GGA ACT TCT CCT GGT CTC
  Asn Asn Ser Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gln Thr Ser Pro Gly Leu
        2060         2070         2080          2090         2100         2110
          *            *            *             *            *            *
  TCA GCT GGG GCC ACT GTC GGC ATC ATG ATT GGA GTG CTG GTT GGG GTT GCT CTG ATA
  Ser Ala Gly Ala Thr Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile
            2120         2130         2140          2150         2160
              *            *            *             *            *
  TAG CAG CCC TGC TCT AGT TGT TGG ATT TCA GGA AAA CTG ACA GTC GTT TTG CTT CTT
  2170          2180         2170         2200          2210         2220
    *             *            *            *             *            *
  CCT TAA AGC ATT TGC AAC AGC TAC AGT CTA AAA TTG CTT CTT TAC CAA GGA TAT TTA
      2230          2240         2250         2260          2270         2280
        *             *            *            *             *            *
  CAG AAA ATA CTC TGA CCA GAG ATC GAG ACC ATC CTA GCC AAC ATC GTG AAA CCC CAT
          2290          2300         2310         2320          2330
            *             *            *            *             *
  CTC TAC TAA AAA TAC AAA AAT GAG CTG GGC TTG GTG GCG CGC ACC TGT AGT CCC AGT
 2340         2350          2360         2370         2380          2390
   *            *             *            *            *             *
  TAC TCG GGA GGC TGA GGC AGG AGA ATC GCT TGA ACC CGG GAG GTG GAG ATT GCA GTG
     2400         2410          2420         2430         2440          2450
       *            *             *            *            *             *
  AGC CCA GAT CGC ACC ACT GCA CTC CAG TCT GGC AAC AGA GCA AGA CTC CAT CTC AAA
         2460         2470          2480         2490         2500
           *            *             *            *            *
  AAG AAA AGA AAA GAA GAC TCT GAC CTG TAC TCT TGA ATA CAA GTT TCT GAT ACC ACT
2510         2520         2530          2540         2550         2560
  *            *            *             *            *            *
  GCA CTG TCT GAG AAT TTC CAA AAC TTT AAT GAA CTA ACT GAC AGC TTC ATG AAA CTG
    2570         2580         2590          2600         2610         2620
      *            *            *             *            *            *
  TCC ACC AAG ATC AAG CAG AGA AAA TAA TTA ATT TCA TGG GGA CTA AAT GAA CTA ATG
        2630         2640         2650          2660         2670         2680
          *            *            *             *            *            *
  AGG ATA ATA TTT TCA TAA TTT TTT ATT TGA AAT TTT GCT GAT TCT TTA AAT GTC TTG
            2690         2700         2710          2720         2730
              *            *            *             *            *
  TTT CCC AGA TTT CAG GAA ACT TTT TTT CTT TTA AGC TAT CCA CTC TTA CAG CAA TTT
  2740          2750         2760         2770          2780         2790
    *             *            *            *             *            *
  GAT AAA ATA TAC TTT TGT GAA CAA AAA TTG AAA CAT TTA CAT TTT ATC CCT ATG TGG
      2800          2810         2820         2830
        *             *            *            *
  TCG CTC CAG ACT TGG GAA ACT ATT CAT GAA TAT TTA TAT TGT ATG
CEA-(c):
        10                  30                  50
         .         .         .         .         .         .
CAGCCGTGCTCGAAGCGTTCCTGGAGCCCAAGCTCTCCTCCACAGGTGAAGACAGGGCCA
        70                  90                 110
         .         .         .         .         .         .
GCAGGAGACACCATGGGGCACCTCTCAGCCCCACTTCACAGAGTGCGTGTACCCTGGCAG
            MetGlyHisLeuSerAlaProLeuHisArgValArgValProTrpGln
       130                 150                 170
         .         .         .         .         .         .
GGGCTTCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCCAGCTC
GlyLeuLeuLeuThrAlaSerLeuLeuThrPheTrpAsnProProThrThrAlaGlnLeu
       190                 210                 230
         .         .         .         .         .         .
ACTACTGAATCCATGCCATTCAATGTTGCAGAGGGGAAGGAGGTTCTTCTCCTTGTCCAC
ThrThrGluSerMetProPheAsnValAlaGluGlyLysGluValLeuLeuLeuValHis
       250                 270                 290
         .         .         .         .         .         .
AATCTGCCCCAGCAACTTTTTGGCTACAGCTGGTACAAAGGGGAAAGAGTGGATGGCAAC
AsnLeuProGlnGlnLeuPheGlyTyrSerTrpTyrLysGlyGluArgValAspGlyAsn
       310                 330                 350
         .         .         .         .         .         .
CGTCAAATTGTAGGATATGCAATAGGAACTCAACAAGCTACCCCAGGGCCCGCAAACAGC
ArgGlnIleValGlyTyrAlaIleGlyThrGlnGlnAlaThrProGlyProAlaAsnSer
       370                 390                 410
         .         .         .         .         .         .
GGTCGAGAGACAATATACCCCAATGCATCCCTGCTGATCCAGAACGTCACCCAGAATGAC
GlyArgGluThrIleTyrProAsnAlaSerLeuLeuIleGlnAsnValThrGlnAsnAsp
       430                 450                 470
         .         .         .         .         .         .
ACAGGATTCTACACCCTACAAGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGA
ThrGlyPheTyrThrLeuGlnValIleLysSerAspLeuValAsnGluGluAlaThrGly
       490                 510                 530
         .         .         .         .         .         .
CAGTTCCATGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAACCCT
GlnPheHisValTyrProGluLeuProLysProSerIleSerSerAsnAsnSerAsnPro
       550                 570                 590
         .         .         .         .         .         .
GTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACACAACCTAC
ValGluAspLysAspAlaValAlaPheThrCysGluProGluThrGlnAspThrThrTyr
       610                 630                 650
         .         .         .         .         .         .
CTGTGGTGGATAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGC
LeuTrpTrpIleAsnAsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnGly
       670                 690                 710
         .         .         .         .         .         .
AACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGACACAGGACCCTATGAGTGTGAA
AsnArgThrLeuThrLeuLeuSerValThrArgAsnAspThrGlyProTyrGluCysGlu
       730                 750                 770
         .         .         .         .         .         .
ATACAGAACCCAGTGAGTGCGAACCGCAGTGACCCAGTCACCTTGAATGTCACCTATGGC
IleGlnAsnProValSerAlaAsnArgSerAspProValThrLeuAsnValThrTyrGly
       790                 810                 830
         .         .         .         .         .         .
CCGGACACCCCCACCATTTCCCCTTCAGACACCTATTACCGTCCAGGGGCAAACCTCAGC
ProAspThrProThrIleSerProSerAspThrTyrTyrArgProGlyAlaAsnLeuSer
       850                 870                 890
         .         .         .         .         .         .
CTCTCCTGCTATGCAGCCTCTAACCCACCTGCACAGTACTCCTGGCTTATCAATGGAACA
LeuSerCysTyrAlaAlaSerAsnProProAlaGlnTyrSerTrpLeuIleAsnGlyThr
       910                 930                 950
         .         .         .         .         .         .
TTCCAGCAAAGCACACAAGAGCTCTTTATCCCTAACATCACTGTGAATAATAGTGGATCC
PheGlnGlnSerThrGlnGluLeuPheIleProAsnIleThrValAsnAsnSerGlySer
       970                 990                1010
         .         .         .         .         .         .
TATACCTGCCACGCCAATAACTCAGTCACTGGCTGCAACAGGACCACAGTCAAGACGATC
TyrThrCysHisAlaAsnAsnSerValThrGlyCysAsnArgThrThrValLysThrIle
      1030                1050                1070
         .         .         .         .         .         .
ATAGTCACTGAGCTAAGTCCAGTAGTAGCAAAGCCCCAAATCAAAGCCAGCAAGACCACA
IleValThrGluLeuSerProValValAlaLysProGlnIleLysAlaSerLysThrThr
      1090                1110                1130
         .         .         .         .         .         .
GTCACAGGAGATAAGGACTCTGTGAACCTGACCTGCTCCACAAATGACACTGGAATCTCC
ValThrGlyAspLysAspSerValAsnLeuThrCysSerThrAsnAspThrGlyIleSer
      1150                1170                1190
         .         .         .         .         .         .
ATCCGTTGGTTCTTCAAAAACCAGAGTCTCCCGTCCTCGGAGAGGATGAAGCTGTCCCAG
IleArgTrpPhePheLysAsnGlnSerLeuProSerSerGluArgMetLysLeuSerGln
      1210                1230                1250
         .         .         .         .         .         .
GGCAACACCACCCTCAGCATAAACCCTGTCAAGAGGGAGGATGCTGGGACGTATTGGTGT
GlyAsnThrThrLeuSerIleAsnProValLysArgGluAspAlaGlyThrTyrTrpCys
      1270                1290                1310
         .         .         .         .         .         .
GAGGTCTTCAACCCAATCAGTAAGAACCAAAGCGACCCCATCATGCTGAACGTAAACTAT
GluValPheAsnProIleSerLysAsnGlnSerAspProIleMetLeuAsnValAsnTyr
      1330                1350                1370
         .         .         .         .         .         .
AATGCTCTACCACAAGAAAATGGCCTCTCACCTGGGGCCATTGCTGGCATTGTGATTGGA
AsnAlaLeuProGlnGluAsnGlyLeuSerProGlyAlaIleAlaGlyIleValIleGly
      1390                1410                1430
         .         .         .         .         .         .
GTAGTGGCCCTGGTTGCTCTGATAGCAGTAGCCCTGGCATGTTTTCTGCATTTCGGGAAG
ValValAlaLeuValAlaLeuIleAlaValAlaLeuAlaCysPheLeuHisPheGlyLys
      1450                1470                1490
         .         .         .         .         .         .
ACCGGCAGGGCAAGCGACCAGCGTGATCTCACAGAGCACAAACCCTCAGTCTCCAACCAC
ThrGlyArgAlaSerAspGlnArgAspLeuThrGluHisLysProSerValSerAsnHis
      1510                1530                1550
         .         .         .         .         .         .
ACTCAGGACCACTCCAATGACCCACCTAACAAGATGAATGAAGTTACTTATTCTACCCTG
ThrGlnAspHisSerAsnAspProProAsnLysMetAsnGluValThrTyrSerThrLeu
      1570                1590                1610
         .         .         .         .         .         .
AACTTTGAAGCCCAGCAACCCACACAACCAACTTCAGCCTCCCCATCCCTAACAGCCACA
AsnPheGluAlaGlnGlnProThrGlnProThrSerAlaSerProSerLeuThrAlaThr
      1630                1650                1670
         .         .         .         .         .         .
GAAATAATTTATTCAGAAGTAAAAAAGCAGTAATGAAACCTGTCCTGCTCACTGCAGTGC
GluIleIleTyrSerGluValLysLysGln
      1690                1710                1730
         .         .         .         .         .         .
TGATGTATTTCAAGTCTCTCACCCTCATCACTAGGAGATTCCTTTCCCCTGTAGGGTAGA
      1750                1770                1790
         .         .         .         .         .         .
GGGGTGGGGACAGAAACAACTTTCTCCTACTCTTCCTTCCTAATAGGCATCTCCAGGCTG
      1810                1830                1850
         .         .         .         .         .         .
CCTGGTCACTGCCCCTCTCTCAGTGTCAATAGATGAAAGTACATTGGGAGTCTGTAGGAA
      1870                1890                1910
         .         .         .         .         .         .
ACCCAACCTTCTTGTCATTGAAATTTGGCAAAGCTGACTTTGGGAAAGAGGGACCAGAAC
      1930                1950                1970
         .         .         .         .         .         .
TTCCCCTCCCTTCCCCTTTTCCCAACCTGGACTTGTTTTAAACTTGCCTGTTCAGAGCAC
      1990                2010                2030
         .         .         .         .         .         .
TCATTCCTTCCCACCCCCAGTCCTGTCCTATCACTCTAATTCGGATTTGCCATAGCCTTG
      2050                2070                2090
         .         .         .         .         .         .
AGGTTATGTCCTTTTCCATTAAGTACATGTGCCAGGAAACAGCGAGAGAGAGAAAGTAAA
      2110                2130                2150
         .         .         .         .         .         .
CGGCAGTAATGCTTCTCCTATTTCTCCAAAGCCTTGTGTGAACTAGCAAAGAGAAGAAAA
      2170                2190                2210
         .         .         .         .         .         .
TCAAATATATAACCAATAGTGAAATGCCACAGGTTTGTCCACTGTCAGGGTTGTCTACCT
      2230                2250                2270
         .         .         .         .         .         .
GTAGGATCAGGGTCTAAGCACCTTGGTGCTTAGCTAGAATACCACCTAATCCTTCTGGCA
      2290                2310                2330
         .         .         .         .         .         .
AGCCTGTCTTCAGAGAACCCACTAGAAGCAACTAGGAAAAATCACTTGCCAAAATCCAAG
      2350                2370                2390
         .         .         .         .         .         .
GCAATTCCTGATGGAAAATGCAAAAGCACATATATGTTTTAATATCTTTATGGGCTCTGT
      2410                2430                2450
         .         .         .         .         .         .
TCAAGGCAGTGCTGAGAGGGAGGGGTTATAGCTTCAGGAGGGAACCAGCTTCTGATAAAC
      2470                2490                2510
         .         .         .         .         .         .
ACAATCTGCTAGGAACTTGGGAAAGGAATCAGAGAGCTGCCCTTCAGCGATTATTTAAAT
      2530                2550                2570
         .         .         .         .         .         .
TGTTAAAGAATACACAATTTGGGGTATTGGGATTTTTCTCCTTTTCTCTGAGACATTCCA
      2590                2610                2630
         .         .         .         .         .         .
CCATTTTAATTTTTGTAACTGCTTATTTATGTGAAAAGGGTTATTTTTACTTAGCTTAGC
      2650                2670                2690
         .         .         .         .         .         .
TATGTCAGCCAATCCGATTGCCTTAGGTGAAAGAAACCACCGAAATCCCTCAGGTCCCTT
      2710                2730                2750
         .         .         .         .         .         .
GGTCAGGAGCCTCTCAAGATTTTTTTTGTCAGAGGCTCCAAATAGAAAATAAGAAAAGGT
      2770                2790                2810
         .         .         .         .         .         .
TTTCTTCATTCATGGCTAGAGCTAGATTTAACTCAGTTTCTAGGCACCTCAGACCAATCA
      2830                2850                2870
         .         .         .         .         .         .
TCAACTACCATTCTATTCCATGTTTGCACCTGTGCATTTTCTGTTTGCCCCCATTCACTT
      2890                2910                2930
         .         .         .         .         .         .
TGTCAGGAAACCTTGGCCTCTGCTAAGGTGTATTTGGTCCTTGAGAAGTGGGAGCACCCT
      2950                2970                2990
         .         .         .         .         .         .
ACAGGGACACTATCACTCATGCTGGTGGCATTGTTTACAGCTAGAAAGCTGCACTGGTGC
      3010                3030                3050
         .         .         .         .         .         .
TAATGCCCCTTGGGAAATGGGGCTGTGAGGAGGAGGATTATAACTTAGGCCTAGCCTCTT
      3070                3090                3110
         .         .         .         .         .         .
TTAACAGCCTCTGAAATTTATCTTTTCTTCTATGGGGTCTATAAATGTATCTTATAATAA
      3130                3150                3170
         .         .         .         .         .         .
AAAGGAAGGACAGGAGGAAGACAGGCAAATGTACTTCTCACCCAGTCTTCTACACAGATG
      3190                3210                3230
         .         .         .         .         .         .
GAATCTCTTTGGGGCTAAGAGAAAGGTTTTATTCTATATTGCTTACCTGATCTCATGTTA
      3250                3270                3290
         .         .         .         .         .         .
GGCCTAAGAGGCTTTCTCCAGGAGGATTAGCTTGGAGTTCTCTATACTCAGGTACCTCTT
      3310                3330                3350
         .         .         .         .         .         .
TCAGGGTTTTCTAACCCTGACACGGACTGTGCATACTTTCCCTCATCCATGCTGTGCTGT
      3370                3390                3410
         .         .         .         .         .         .
GTTATTTAATTTTTCCTGGCTAAGATCATGTCTGAATTATGTATGAAAATTATTCTATGT
      3430                3450
         .         .         .         .         
TTTTATAATAAAAATAATATATCAGACATCGAAAAAAAAAA
CEA-(d):
            10           20            30           40           50
            |            |             |            |            |
CC GGG GGA CAC GCA GGG CCA ACA GTC ACA GCA GCC CTG ACC AGA GCA TTC CTG GAG CTC
   60           70           80            90          100         110
   |           |           |             |            |            |
   AAG CTC TCT ACA AAG AGG TCG ACA GAG AAG ACA GCA GAG ACC ATG GGA CCC CCC TCA
                                                           Mel Gly Pro Pro Ser
      120          130          140           150          160          170
       |            |            |             |            |            |
   GCC CCT CCC TGC AGA TTG CAT GTC CCC TGG AAG GAG GTC CTG CTC ACA GCC TCA CTT
   Ala Pro Pro Cys Arg Leu His Val Pro Trp Lys Glu Val Leu Leu Thr Ala Ser Leu
          180          190          200           210          220          230
           |            |            |             |            |            |
   CTA ACC TTC TGG AAC CCA CCC ACC ACT GCC AAG CTC ACT ATT GAA TCC ACG CCA TTC
   Leu Thr Phe Trp Asn Pro Pro Thr Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe
              240          250          260           270          280
               |            |            |             |            |
   AAT GTC GCA GAG GGG AAG GAG GTT CTT CTA CTC GCC CAC AAC CTG CCC CAG AAT CGT
   Asn Val Ala Glu Gly Lys Glu Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg
    290           300          310          320           330          340
     |             |            |            |             |            |
   ATT GGT TAC AGC TGG TAC AAA GGC GAA AGA GTG GAT GGC AAC AGT CTA ATT GTA GGA
   Ile Gly Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly
        350           360          370          380           390          400
         |             |            |            |             |            |
   TAT GTA ATA GGA ACT CAA CAA GCT ACC CCA GGG CCC GCA TAC AGT GGT CGA GAG ACA
   Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg Glu Thr
            410           420          430          440           450
             |             |            |            |             |
   ATA TAC CCC AAT GCA TCC CTG CTG ATC CAG AAC GTC ACC CAG AAT GAC ACA GGA TTC
   Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln Asn Asp Thr Gly Phe
   
   460          470           480          490          500           510
    |            |             |            |            |             |
   TAC ACC CTA CAA GTC ATA AAG TCA GAT CTT GTG AAT GAA GAA GCA ACC GGA CAG TTC
   Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val Asn Glu Glu Ala Thr Gly Gln Phe
       520          530           540          550          560           570
        |            |             |            |            |             |
   CAT GTA TAC CCG GAG CTG CCC AAG CCC TCC ATC TCC AGC AAC AAC TCC AAC CCC GTG
   His Val Tyr Pro Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val
           580          590           600          610          620
            |            |             |            |            |
   GAG GAC AAG GAT GCT GTG GCC TTC ACC TGT GAA CCT GAG GTT CAG AAC ACA ACC TAC
   Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Val Gln Asn Thr Thr Tyr
   630         640          650           660          670          680
   |            |            |             |            |            |
   CTG TGG TGG GTA AAT GGT CAG AGC CTC CCG GTC AGT CCC AGG CTG CAG CTG TCC AAT
   Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser Asn
      690          700          710           720          730          740
       |            |            |             |            |            |
   GGC AAC AGG ACC CTC ACT CTA CTC AGC GTC AAA AGG AAC GAT GCA GGA TCG TAT GAA
   Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Lys Arg Asn Asp Ala Gly Ser Tyr Glu
          750          760          770           780          790          800
           |            |            |             |            |            |
   TGT GAA ATA CAG AAC CCA GAG AGT GCC AAC CGC AGT GAC CCA GTC ACC CTG AAT GTC
   Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asn Val
              810          820          830           840          850
               |            |            |             |            |
   CTC TCT GGC CCA GAT GGC CCG ACC ATT TCC CCC TCA AAG GCC AAT TAC CGT CCA GGG
   Leu Tyr Gly Pro Asp Gly Pro Thr Ile Ser Pro Ser Lys Ala Asn Tyr Arg Pro Gly
    860           870          880          890           900          910
     |             |            |            |             |            |
   GAA AAT CTG AAC CTC TCC TGC CAC GCA GCC TCT AAC CCA CCT GCA CAG TAC TCT TGG
   Glu Asn Leu Asn Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp
        920          930          940          950           960          970
         |             |            |            |             |            |
   TTT ATC AAT GGG ACG TTC CAG CAA TCC ACA CAA GAG CTC TTT ATC CCC AAC ATC ACT
   Phe Ile Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr
            980           990         1000         1010          1020
             |             |            |            |             |
   GTG AAT AAT AGC GAA TCC TCT ATG TGC CAA GCC CAT AAC TCA GCC ACT GGC CTC AAT
   Val Asn Asn Ser Gly Ser Tyr Kel Cys Gln Ala His Asn Ser Ala Thr Gly Leu Asn
   1030         1040          1050         1060         1070          1080
    |            |             |            |            |             |
   AGG ACC ACA GTC ACG ATG ATC ACA GTC TCT GGA AGT GCT CCT GTC CTC TCA GCT GTG
   Arg Thr Thr Val Thr Kel Ile Thr Val Ser Gly Ser Ala Pro Val Leu Ser Ala Val
      1090         1100          1110         1120         1130          1140
        |            |             |            |            |             |
   GCC ACC GTC GGC ATC ACG ATT GGA GTG CTG GCC AGG GTG GCT CTG ATA TAG CAG CCC
   Ala Thr Val Gly Ile Thr Ile Gly Val Try Ala Arg Val Ala Leu Ile ...
          1150         1160          1170         1180         1190
            |            |             |            |            |
   TGG TGT ATT TTC GAT ATT TCA GGA AGA CTG GCA GAT TGG ACC AGA CCC TGA ATT CTT
   1200       1210         1220          1230         1240         1250
     |          |            |             |            |            |
   CTA GCT CCT CCA ATC CCA TTT TAT CCC ATG GAA CCA CTA AAA ACA AGG TCT GCT CTG
     1260         1270         1280          1290         1300         1310
       |            |            |             |            |            |
   CTC CTG AAG CCC TAT ATG CTG GAG ATG GAC AAC TCA ATG AAA ATT TAA AGG AAA AAC
         1320         1330         1340          1350         1360         1370
           |            |            |             |            |            |
   CCT CAG GCC TGA GGT GTG TGC CAC TCA GAG ACT TCA CCT AAC TAG AGA CAG GCA AAC
             1380         1390         1400          1410         1420
               |            |            |             |            |
   TGC AAA CCA AAC CTC TTT CGC TTG GCA GGA TGA TGG TGT CAT TAG TAT TTC ACA AGA
   1430          1440         1450         1460          1470         1480
     |             |            |            |             |            |
   AGT AGC TTC AGA GGG TAA CTT AAC AGA GTA TCA GAT CTA TCT TGT CAA TCC CAA CGT
       1490          1500         1510         1520          1530         1540
         |             |            |            |             |            |
   TTT ACA TAA AAT AAG CGA TCC TTT AGT GCA CCC AGT GAC TGA CAT TAG CAG CAT CTT
          1550          1560          1570         1580          1590
             |             |            |            |             |
   TAA CAC AGC CGT GTG TTC AAG TGT ACA GTG GTC CTT TTC AGA GTT GGA AAT ACT CCA
   1600         1610          1620         1630         1640          1650
    |            |             |            |            |             |
   ACT GAA ATG TTA AGG AAG AAG ATA GAT CCA ATT AAA AAA AAT TAA AAC CAA TTT AAA
      1660         1670          1680         1690         1700          1710
        |            |             |            |            |             |
   AAA AAA AAA GAA CAC AGG AGA TTC CAG TCT ACT TGA GTT AGC ATA ATA CAG AAG TCC
          1720         1730          1740         1750         1760
            |            |             |            |            |
   CCT CTA CTT TAA CTT TTA CAA AAA AGT AAC CTG AAC TAA TCT GAT GTT AAC CAA TGT
   1770       1780         1790          1800         1810         1820
     |          |            |             |            |            |
   ATT TAT TTG TCT GGT TCT GTT TCC TTG TTC CAG TTT GAC AAA ACC CAC TGT TCT TGT
     1830         1840         1850          1860         1870         1880
       |            |            |             |            |            |
   ATT GTA TTG CCC AGG GGG AGC TAT CAC TGT ACT TGT ACA GTG GTG CTG CTT TAA GTT
         1890         1900         1910          1920         1930         1940
           |            |            |             |            |            |
   CAT AAA TCA CAA ATA AAA GCC AAT TAG CTC TAT AAC TAA AAA AAA AAA AAA AAA AAA
             1950         1960
               |            |
   AAA AAA AAA AAA AAA AAA AAA AAA
A schematic relationship of the transmembrane CEA's, namely TM-1 (CEA-(c)), TM-2 (CEA-(e)), TM-3 (CEA-(f)) and TM-4 (CEA-(g)) is depicted in FIG. 1:
Assuming TM-1 is composed of five sections as depicted in FIG. 1, namely 10, 12, 14, 16 and 18, TM-2 differs from TM-1 in that the 100 amino acids (100 AA) section 14 is deleted and at splice point 20 between sections 12 and 16, surprisingly an extra amino acid, namely Asp occurs.
TM-3 is the same as TM-1 except that section 18 is truncated at splice point 22, i.e., a section of 70 amino acids is deleted and results in a new section made up of subsections 24+26. Surprisingly, however, six new amino acids (section 26) occur. Another example of formation of a novel amino acid sequences relating from a deletion of nucleic acid sequence is for platelet derived growth factor-A.
TM-4 is the same as TM-2 up until the end of subsection 24.
Subsection 24 is contained in section 18 of TM-1 and TM-2, but is not depicted in FIG. 1 for TM-1 and TM-2.
Some CEA epitopes are unique. These are the epitopes which have been useful for distinguishing the various CEA-like antigens immunologically. Peptide epitopes are defined by the linear amino acid sequence of the antigen and/or features resulting from protein folding. The information required for protein following is encoded in the primary amino acid sequence. Therefore, antigenic differences ultimately result from differences in the primary structure of the difference CEA molecules. The differences residing in the CEA protein in the CEA species can thus be determined by determining the primary amino acid sequences. This can be most readily accomplished by cloning and sequencing each of the genes for CEA. To determine which gene products will be most useful for cancer diagnosis, unique probes can be selected for each gene and expression of each gene can be determined in different tumor types by nucleic acid hybridization techniques. The present invention provides a tool with which to identify potential genes coding for different members of the CEA family and to determine the theoretical primary amino acid sequences for them. Using the method of automated peptide synthesis, peptides can then be synthesized corresponding to unique sequences in these antigens. With these peptides, antibodies to these sequences can be produced which, in the intact CEA molecule, might not be recognized by the animal being immunized. Having accomplished this, advantage can then be taken of the differences in these antigens to generate specific immunoassays for the measurement of each antigen.
A wide variety of host/cloning vehicle combinations may be employed in cloning the double-stranded nucleic acid prepared in accordance with this invention. For example, useful cloning vehicles may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., plasmids from E. coli including col E1, pCR1, pBR322, pMB89 and their derivatives, wider host range plasmids, e.g., RP4, and phage DNAs, e.g., the numerous derivatives of phage, e.g., NM989, and other DNA phages, e.g., M13 and Filamenteous single-stranded DNA phages and vectors derived from combinations of plasmids and phage DNAs such as plasmids which have been modified to employ phage DNA or other expression control sequences or yeast plasmids such as the 2μ plasmids or derivatives thereof. Useful hosts may include bacterial hosts such as strains of E. coli, such as E. coli HB 101, E. coli X1776, E. coli X2282, E. coli MRC1 and strains of Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus and other E. coli, bacilli, yeasts and other fungi, animal or plant hosts such as animal (including human) or plant cells in culture or other hosts. Of course, not all hosts/vector combinations may be equally efficient. The particular selection of host/cloning vehicle combination may be made by those of skill in the art after due consideration of the principles set forth without departing from the scope of this invention.
Furthermore, within each specific cloning vehicle, various sites may be selected for insertion of the nucleic acid according to the present invention. These sites are usually designated by the restriction endonuclease which cuts them. For example, in pBR322 the Pst1 site is located in the gene for beta-lactamase, between the nucleotide triplets that code for amino acids 181 and 182 of that protein. One of the two HindII endonuclease recognition sites is between the triplets coding for amino acids 101 and 102 and one of the several Taq sites at the triplet coding for amino acid 45 of beta-lactamase in pBR322. In similar fashion, the EcoRI site and the PVUII site in this plasmid lie outside of any coding region, the EcoR1 site being located between the genes coding for resistance to tetracycline and ampicillin, respectively. These sites are well recognized by those of skill in the art. It is, of course, to be understood that a cloning vehicle useful in this invention need not have a restriction endonuclease site for insertion of the chosen DNA fragment. Instead, the vehicle could be cut and joined to the fragment by alternative means.
The vector or cloning vehicle and in particular the site chosen therein for attachment of a selected nucleic acid fragment to form a recombinant nucleic acid molecule is determined by a variety of factors, e.g., the number of sites susceptible to a particular restriction enzyme, the size of the protein to be expressed, the susceptibility of the desired protein to proteolytic degradation by host cell enzymes, the contamination of the protein to be expressed by host cell proteins difficult to remove during purification, the expression characteristics, such as the location of start and stop codons relative to the vector sequences, and other factors recognized by those of skill in the art. The choice of a vector and an insertion site for a particular gene is determined by a balance of these factors, not all sections being equally effective for a given case.
Methods of inserting nucleic acid sequences into cloning vehicles to form recombinant nucleic acid molecules include, for example, dA-dT tailing, direct ligation, synthetic linkers, exonuclease and polymerase-linked repair reactions followed by ligation, or extension of the nucleic acid strand with an appropriate polymerase and an appropriate single-stranded template followed by ligation.
It should also be understood that the nucleotide sequences or nucleic acid fragments inserted at the selected site of the cloning vehicle may include nucletodies which are not part of the actual structural gene for the desired polypeptide or mature protein or may include only a fragment of the complete structural gene for the desired protein or mature protein.
The cloning vehicle or vector containing the foreign gene is employed to transform an appropriate host so as to permit that host to replicate the foreign gene and to express the protein coded by the foreign gene or portion thereof. The selection of an appropriate host is also controlled by a number of factors recognized by the art. These include, for example, the compatibility with the chosen vector, the toxicity of proteins encoded by the hybrid plasmid, the ease of recovery of the desired protein, the expression characteristics, biosafety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for expression of a particular recombinant DNA molecule.
The level of production of a protein is governed by two major factors: the number of copies of its gene within the cell and the efficiency with which those gene copies are transcribed and translated. Efficiency of transcription and translation (which together comprise expression) is in turn dependent upon nucleotide sequences, normally situated ahead of the desired coding sequence. These nucleotide sequences or expression control sequences define inter alia, the location at which RNA polymerase interacts to initiate transcription (the promoter sequence) and at which ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation. Not all such expression control sequences function with equal efficiency. It is thus of advantage to separate the specific coding sequences for the desired protein from their adjacent nucleotide sequences and fuse them instead to other known expression control sequences as as to favor higher levels of expression. This having been achieved, the newly engineered nucleic acid, e.g., DNA, fragment may be inserted into a multicopy plasmid or a bacteriophage derivative in order to increase the number of gene copies within the cell and thereby further improve the yield of expressed protein.
Several expression control sequences may be employed as described above. These include the operator, promoter and robosome binding and interaction sequences (including sequences such as the Shine-Dalgarno sequences) of the lactose operon of E. coli (“the lac system”), the corresponding sequences of the tryptophan synthetase system of E. coli (“the trp system”), the major operator and promoter regions of phage λ (OLPL and ORP′R), the control region of Filamenteous single-stranded DNA phages, or other sequences which control the expression of genes of prokaryotic or eukaryotic cells and their viruses. Therefore, to improve the production of a particular polypeptide in an appropriate host, the gene coding for that polypeptide may be selected and removed from a recombinant nucleic acid molecule containing it and reinserted into a recombinant nucleic acid molecule closer or in a more appropriate relationship to its former expression control sequence or under the control of one of the above described expression control sequences. Such methods are known in the art.
as used herein “relationship” may encompass many factors, e.g., the distance separating the expression enhancing and promoting regions of the recombinant nucleic acid molecule and the inserted nucleic acid sequence, the transcription and translation characteristics of the inserted nucleic acid sequence or other sequences in the vector itself, the particular nucleotide sequence of the inserted nucleic acid sequence and other sequences of the vector and the particular characteristics of the expression enhancing and promoting regions of the vector.
Further increases in the cellular yield of the desired products depend upon an increase in the number of genes that can be utilized in the cell. This is achieved, for illustration purposes, by insertion of recombinant nucleic acid molecules engineered into the temperate bacteriophage λ (NM989), most simply by digestion of the plasmid with a restriction enzyme, to give a linear molecule which is then mixed with a restricted phage λ cloning vehicle (e.g., of the type described by N. E. Murray et al, “Lambdoid Phages That Simplify the Recovery of In Vitro Recombinants”, Molec. Gen. Genet., 150, pp. 53-61 (1977) and N. E. Murray et al, “Molecular Cloning of the DNA Ligase Gene From Bacteriophage T4”, J. Mol. Biol., 132, pp. 493-505 (1979)) and the recombinant DNA molecule recircularized by incubation with DNA ligase. The desired recombinant phage is then selected as before and used to lysogenize a host strain of E. coli.
Particularly useful λ cloning vehicles contain a temperature-sensitive mutation in the repression gene cl and suppressible mutations in gene S, the product of which is necessary for lysis of the host cell, and gene E, the product of which is major capsid protein of the virus. With this system, the lysogenic cells are grown at 32° C. and then heated to 45° C. to induce excision of the prophage. Prolonged growth at 37° C. leads to high levels of production of the protein, which is retained within the cells, since these are not lysed by phage gene products in the normal way, and since the phage gene insert is not encapsulated it remains available for further transcription. Artificial lysis of the cells then releases the desired product in high yield.
In addition, it should be understood that the yield of polypeptides prepared in accordance with this invention may also be improved by substituting different codons for some or all of the codons of the present DNA sequences, these substituted codons coding for amino acids identical to those codes for by the codons replaced.
Finally, the activity of the polypeptides produced by the recombinant nucleic acid molecules of this invention may be improved by fragmenting, modifying or derivatizing the nucleic acid sequences or polypeptides of this invention by well-known means, without departing from the scope of this invention.
The polypeptides of the present invention include the following:
(1) the polypeptides expressed by the above described cells,
(2) polypeptides prepared by synthetic means,
(3) fragments of polypeptides (1) or (2) above, such fragments produced by synthesis of amino acids or by digestion or cleavage.
Regarding the synthetic peptides according to the invention, chemical synthesis of peptides is described in the following publications: S. B. H. Kent, Biomedical Polymers, eds. Goldberg, E. P. and Nakajima, A. (Academic Press, New York), 213-242, (1980); A. R. Mitchell, S. B. H. Kent, M. Engelhard and R. B. Merrifield, J. Org. Chem., 43, 2845-2852, (1978); J. P. Tam, T. -W. Wong, M. Riemen, F. -S. Tjoeng and R. B. Merrifield, Tet. Letters, 4033-4036, (1979); S. Mojsov, A. R. Mitchell and R. B. Merrifield, J. Org. Chem., 45, 555-560, (1980); J. P. Tam, R. D. DiMarchi and R. B. Merrifield, Tet. Letters, 2851-2854, (1981); and S. B. H. Kent, M. Riemen, M. Le Doux and R. B. Merrifield, Proceedings of the IV International Symposium on Methods of Protein Sequence Analysis, (Brookhaven Press, Brookhaven, NY), in press, 1981.
In the Merrifield solid phase procedure, the appropriate sequence of L-amino acids is built up form the carboxyl terminal amino acid to the amino terminal amino acid. Starting with the appropriate carboxyl terminal amino acid attached to a polystyrene (or other appropriate) resin via chemical linkage to a chloromethyl group, benzhydrlamine group, or other reactive group of the resin, amino acids are added one by one using the following procedure. The peptide-resin is:
(a) washed with methylene chloride;
(b) neutralized by making for 10 minutes at room temperature with 5% (v/v) diisopropylethylamine (or other hindered base) in methylene chloride;
(c) washed with methylene chloride;
(d) an amount of amino acid equal to six times the molar amount of the growing peptide chain is activated by combining it with one-half as many moles of a carbodiimide (e.g., dicyclohexylcarbodiimide, or diiospropylcarbodiimide) for ten minutes at 0° C., to form the symmetric anhydride of the amino acid. The amino acid used should be provided originally as the N-alpha-tert.-butyloxycarbonyl derivative, with side chains protected with benzyl esters (e.g., aspartic or glutamic acids), benzyl ethers (e.g., serine, threonine, cysteine or tyrosine), benzyloxcarbonyl groups (e.g, lysine) or other protecting groups commonly used in peptide synthesis;
(e) the activated amino acid is reacted with the peptide-resin for two hours at room temperature, resulting in addition of the new amino acid to the end of the growing peptide chain;
(f) the peptide-resin is washed with methylene chloride;
(g) the N-alpha-(tert.-butyloxycarbonyl) group is removed from the most recently added amino acid by reacting with 30 to 65%, preferably 50% (v/v) trifluoroacetic acid in methylene chloride for 10 to 30 minutes at room temperature;
(h) the peptide-resin is washed with methylene chloride;
(i) steps (a) through (h) are repeated until the required peptide sequence has been constructed.
The peptide is then removed from the resin and simultaneoulsy the side-chain protecting groups are removed, by reaction with anhydrous hydrofluoric acid containing 10% v/v of anisole or other suitable (aromatic) scavenger. Subsequently, the peptide can be purified by gel filtration, ion exchange, high pressure liquid chromatography, or other suitable means.
In some cases, chemical synthesis can be carried out without the sold phase resin, in which case the synthetic reactions are performed entirely in solution. The reactions are similar and well known in the art, and the final product is essentially identical.
Digestion of the polypeptide can be accomplished by using proteolytic enzymes, especially those enzymes whose substrate specificity results in cleavage of the polypeptide at sites immediately adjacent to the desired sequence of amino acids.
Cleavage of the polypeptide can be accomplished by chemical means. Particular bonds between amino acids can be cleaved by reaction with specific reagents. Examples include the following: bonds involving methionine are cleaved by cyanogen bromide; asparaginyl-glycine bonds are cleaved by hydroxylamine.
The present invention has the following advantages:
(1) The nucleic acids coding for TM-1, TM-2 and TM-3 can be used as probes to isolate other members of the CEA gene family.
(2) The nucleic acids coding for TM-1, TM-2 and TM-3 can be used to derive oligonucleotide probes to determine the expression of TM-1, TM-2, TM-3 and other CEA genes in various tumor types.
(3) TM-1, TM-2, TM-3 and TM-4 nucleotide sequences can be used to predict the primary amino acid sequence of the protein for production of synthetic peptides.
(4) Synthetic peptides derived from the above sequence can be used to produce sequence-specific antibodies.
(5) Immunoassays for each member of the CEA antigen family can be produced with these sequence-specific antibodies and synthetic peptides.
(6) The aforementioned immunoassays can be used as diagnostics for different types of cancer if it is determined that different members of the CEA family are clinically useful markers for different types of cancer.
Peptides according to the present invention can be labelled by conventional means using radioactive moieties (e.g., 125I), enzymes, dyes and fluorescent compounds, just to name a few.
Several possible configurations for immunoassays according to the present invention can be used. The readout systems capable of being employed in these assays are numerous and non-limiting examples of such systems include fluorescent and colorimetirc enzyme systems, radioisotopic labelling and detection and chemiluminescent systems. Two examples of immunoassay methods are as follows:
(1) An enzyme linked immunoassay (ELISA) using an antibody preparation according to the present invention (including Fab or F(ab)′ fragments derived therefrom) to a solid phase (such as a microtiter plate or latex beads) is attached a purified antibody of a specificity other than that which is conjugated to the enzyme. This solid phase antibody is contacted with the sample containing CEA antigen family members. After washing, the solid phase antibody-antigen complex is contacted with the conjugated anti-peptide antibody (or conjugated fragment). After washing away unbound conjugate, color or fluorescence is developed by adding a chromogenic or fluorogenic substrate for the enzyme. The amount of color or fluorescence developed is proportional to the amount of antigen in the sample.
(2) A competitive fluorometric immunoassay using fluorescently labelled peptide or synthetic peptides of the sequences for TM-2, TM-2, TM-3 and TM-4. In this example, the purified peptide expressed by cells or synthetic peptides thereof are fluorescently labelled. To a solid phase is attached a purified antibody. This solid phase is then contacted with sample containing CEA antigen family members to which has been added fluorescent peptide probe. After binding, excess probe is washed away the amount of bound probe is quantitated. The amount of bound fluorescent probe will be inversely proportional to the amount of antigen in the sample.
In the nucleic acid hybridization method according to the present invention, the nucleic acid probe is conjugated with a label, for example, an enzyme, a fluorophore, a radioisotope, a chemiluminescent compound, etc. In the most general case, the probed would be contacted with the sample and the presence of any hybridizable nucleic acid sequence would be detected by developing in the presence of a chromogenic enzyme substrate, detection of the fluorophore by epifluorescence, by autoradiography of the radioisotopically labelled probed or by chemiluminescence. The detection of hybridizable RNA sequences can be accomplished by (1) a dot blot methodology or (2) an in sutu hybridization methodology. Methods for these last two techniques are described by D. Gillespie and J. Bresser, “mRNA Immobilization in NaI: Quick Blots”, Biotechniques, 184-192, November/December 1983 and J. Lawrence and R. Singer, “Intracellular Localization of Messenger RNAs for Cytosketal Proteins”, Cell, 45, 407-415, May 9, 1986, respectively. The readout systems can be the same as described above, e.g., enzyme labelling, radiolabelling, etc.
As stated above, the invention also relates to the use in medicine of the aforementioned complex of the invention.
The invention further provides a pharmaceutical composition containing as an active ingredient a complex of the invention in the form of a sterile and/or physiologically isotonic aqueous solution.
For parenteral administration, solutions and emulsions containing as an active ingredient the complex of the invention should be sterile and, if appropriate, blood-isotonic.
It is envisaged that the active complex will be administered perorally, parenterally (for example, intramuscularly, intraperitoneally, or intravenously), rectally or locally.
EXAMPLE 1 Preparation of cDNA in pcE22 which codes for TM2-CEA [CEA-(e)] EXAMPLE 1a RNA Preparation
Messenger RNA was prepared by the proteinase K extraction method of J. Favolaro, R. Treisman and R. Kamen, Methods in Enzymology, 65, 718, Academic Press, Inc. (1980), followed by oligo dT cellulose chromatography to yield poly A+ RNA (3′-polyadenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides). To obtain approximately 100 μg of poly A+ RNA, approximately 3×108 cells of transfectant 23.411 (ATCC No. CRL 9731, deposited with the ATCC on Jun. 1, 1988), that expressed TM-1, TM-2, TM-3 and TM-4, Kamarck et al, Proc. Natl. Acad. Sci., USA, 84, 5350-5354, August 1987, were harvested from roller bottles after late logarithmic growth. Cells were lysed by homogenization in an ice-cold solution of 140 mM Nacl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.0, 0.5% NP40, 4 mM dithiothreitol and 20 units of placental ribonuclease inhibitor/ml. sodium deoxycholate was then added to 0.2%. Cytoplasm and nuclei were separated by centrifugation of the homogenate at 12,000 xg for 20 minutes. The cytoplasmic fraction was mixed with an equal volume of 0.2 M tris-HCl, pH 7.8, 23 mM EDTA, 0.3 M NaCl, 2% sodium dodecyl sulfate and 400 μg/ml of proteinase K, incubated for 1 hour at 37° C., then extracted once with an equal volume of phenol/cholorform (1:1/v:v) solution. Nucleic acids were obtained by ethanol precipitation of the separated aqueous phase. Total RNA was enriched by passage in 0.5 M NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% sarcosyl through an oligo dT(12-18) cellulose column. After washing, bound RNA was eluted in the same solution without sodium chloride.
EXAMPLE 1b Reverse Transcription of mRNA
Ten micrograms of poly A+ RNA were primed for reverse transcription with oligo dT(12-18) and pdN6 primers. One hundred microliter reaction was performed for 4 hours at 42° C. with 200 units AMV reverse transcriptase (Life Science, Inc. St. Petersburg, Fla., U.S.A.). The RNA component of the cDNA/mRNA hybrids was replaced with the second complementary strand by treatment with RNase H, E. coli DNA polymerase I and E. coli DNA ligase at 12° C. and 22° C. for 1.5 hours each. Molecular ends were polished by treatment with T4 DNA polymerase. cDNA was phenol/chloroform extracted and purified over a “SEPHADEX G-50” spun column prepared in 10 mM Tris-HCl, ph 7.8, 1 mM EDTA (TE).
EXAMPLE 1c Cloning of pcE22 (plasmid cDNA E22)
Synthetic DNA linkers
5′ pCCCGGG 3′
3′ GGGCCCTTAA 5′
were attached to the ends of cDNA by blunt end ligation with excess T4 DNA ligase. Excess linkers were removed by chromatography through “SEPHADEX G-50” (medium) in TE, and by fractionation on 0.8% low melting agarose gel. Based on Northern blot analysis of poly A+ RNA of the 23.411 cell line, the size of the CEA-related mRNA was estimated at 3.6 kb. Therefore, cDNA fragments between 2 and 4 kb were recovered from gel slices and fragments were ethanol precipiated. After resuspension of cDNA in TE, EcoRI-cleaved lambada gt10 arms were added to cDNA at an estimated molar ration of 1:1. Ligation proceeded at 7° C. for 2 days in the presence of T4 DNA ligase. Aliquots of the ligation reaction were added to commercially-obtained packaging mix (Stratagene, San Diego, Calif. U.S.A.). Five million phage particles were obtained often in vitro packaging and infection of E. coli host NM514.
EXAMPLE 1d Screening of Recombinant Library
Five hundred thousand packaged lambda particles were plated on lawns of E. coli NM514 and replicate patterns were lifted onto nitrocellulose sheets as described by W. D. Benton and R. W. Davis, Science 196, 180-182, (1977). Positive phage were selected by hybridization with 32P-labeled LV7 cDNA insert probe that contained a domain repeated amoung various CEA family members. By multiple rounds of screening. Phage from individual plaques were amplified and titered, and these were used to prepare small quantities of recombinant phage DNA.
EXAMPLE 1e DNA Manipulation
Phage DNA was prepared according to T. Maniatis, E. Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, (1982). DNA segments were isolated from low melting agarose gels and inserted for subcloning into Bluescript plasmid vectors (Stratagene, San Diego, Calif. U.S.A.). DNA sequencing was performed by the dideoxy termination method of F. Sanger, S. Nicklen and A. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74, 5463-5467, (1977). The nucleic acid and translated sequence for cDNA in pcE22 is given hereinabove (TM-2 (CEA-(e)).
EXAMPLE 2 Preparation of cDNA in pcHT-6 which Partically Codes for TM3-CEA [CEA-(f)] EXAMPLE 2a RNA Preparation
Messenger RNA was prepared by the proteinase K extraction method of J. Favolaro, R. Treisman and R. Kamen, Methods in Enzymology, 65 718, Academic Press, Inc. (1980), followed by oligo dT cellulose chromatography to yield poly A+ RNA (3′-poladenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides). To obtain approximately 100 μg of poly A+ RNA, approximately 3×108 cells of HT-29 tumor cells (ATCC HTB38) were harvested form roller bottles after late logarithmic growth. Cells were lysed by homogenization in an ice-cold solution of 140 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.0, 0.5% NP40, 4 mM dithiothreitol and 20 units of placental ribonuclease inhibitor/ml. Sodium deoxycholate was then added to 0.2%. Cytoplasm and nuclei were separated by centriguation of the homogenate at 12,000×g for 20 minutes. The cytoplasmic fraction was mixed with an equal volume of 0.2 M tris-Hcl, pH 7.8, 25 mM EDTA, 0.3 M NaCl, 2% sodium dodecyl sulfate and 400 μg/ml of proteinase K, incubated for 1 hour at 37° C., then extracted once with an equal volume of phenol/choloform (1:1/v:v) solution. Nucleic acids were obtained by ethanol precipitation of the separated aqueous phase. Total RNA was enriched by passage in 0.5 M NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% sarcosyl through an oligo dT(12-18) cellulose column. After washing, bound RNA was eluted in the same solution without sodium chloride.
EXAMPLE 2b Reverse Transcription of mRNA
Ten micrograms of HT-29 poly A+ RNA were primed for reverse transcription with oligo dT(12-18) and pdN6 primers. One hundred microliter reaction was performed for 4 hours at 42° C. with 200 units AMV reverse transcriptase (Life Science, Inc. St. Petersburg, Fla., U.S.A.). The RNA component of the cDNA/mRNA hybrids was replaced with the second complementary strand by treatment with RNase H, E. coli DNA polymerase I and E. coli DNA ligase at 12° C. and 22° C. for 1.5 hours each. Molecular ends were polished by treatment with T4 DNA polymerase. cDNA was phenol/chloroform extracted and purified over a “SEPHADEX G-50” spun column prepared in 10 mM Tris-HCl, pH 7.8, 1 mM EDTA (TE).
EXAMPLE 2c Cloning of pcHT-6 (plasmid cDNA HT-6)
Synthetic DNA linkers
5′ pCCCGGG 3′
3′ GGGCCCTTAA 5′
were attached to the ends of cDNA by blunt end ligation with excess T4 DNA ligase. Excess linkers were removed by chromatography through “SEPHADEX G-50” (medium) in TE, and by fractionation on 0.8% low melting agarose gel. Based on Northern blot analysis of poly A+ RNA of the HT-29 cell line, the size of the CEA-related mRNA was estimated at 2.2 kb. Therefore, cDNA fragments between 2 and 3 kb were recovered from gel slices and fragments were ethanol precipitated. After suspension of cDNA in TE, EcoRI-cleaved lambada gt10 arms were added to cDNA at an estimated molar ratio of 1:1. Ligation proceeded at 7° C. for 2 days in the presence of T4 DNA ligase. Aliquots of the ligation reaction were added to commercially-obtained packaging mix (Stratagene, San Diego, Calif., U.S.A.). Five million phage particles were obtained often in vitro packaging and infection of E. coli host NM514.
EXAMPLE 2d Screening of Recombinant Library
Five hundred thousand packaged lambda particles were plated on lawns of E. coli NM514 and replicate patterns were lifted onto nitrocellulose sheets as described by W. D. Benton and R. W. Davis, Science, 196, 180-182, (1977). Positive phage were selected by hybridization with 32P-labeled LV7 cDNA insert probe that contained a domain repeated amoung various CEA family members. By multiple rounds of screening. Phage from individual plaques were amplified and titered, and these were used to prepare small quantities of recombinant phage DNA.
EXAMPLE 2e DNA Manipulation
Phage DNA was prepared according to T. Maniatis, E. Fitsch and J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Habor, (1982). DNA segments were isolated from low melting agarose gels and inserted for subcloning into Bluescript plasmid vectors (Stratagene, San Diego, Calif., U.S.A.). DNA sequencing was performed by the didexoy termination method of F. Sanger, S. Nicklen and A. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74, 5463-5467, (1977). The nucleic acid and translated sequence for cDNA in HT-6 not complete at the 5′ end of it s coding region, but the nucleotide sequence and restricting map of the HT-6 insert indicates that it is related to nucleic acid sequences of cDNA clones coding for CEA-(c) and CEA-(e). The nucleotide sequence of HT-6 insert differs from these clones at only nucleotide position 1463 to 1515 and 1676 to 2429 of the CEA-(c) cDNA. It is inferred from this result that the pcHT-6 insert is a partial coding sequence for CEA-(f) and the presumed nucleic acid and translated sequence of CEA-(f) is given hereinabove (TM-3 (CEA-(f)).
EXAMPLE 3 Preparation of cDNA which codes for TM4-CEA [CEA-(g)] EXAMPLE 3a RNA Preparation
Messenger RNA is prepared by the proteinase K extraction method of J. Favolaro, R. Treisman and R. Kamen, Methos in Enzymology, 65, 718, Academic Press, Inc. (1980), followed by oligo dT cellulose chromatography to yield poly A+ RNA (3′-polyadenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides). To obtain approximately 100 μg of poly A+ RNA, approximately 3×108 cells of transfectant 23.411 or tumor cell line HT-29 (ATCC HTB 38) are harvested from roller bottles after late logarithmic growth. Cells are lysed by homogenization in an ice-cold solution of 140 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.0, 0.5% NP40, 4 mM dithiothreitol and 20 units of placental ribonuclease inhibitor/ml. Sodium deoxycholate was then added to 0.2%. Cytoplasm and nuclei are separated by centriguation of the homongenate at 12,000×g for 20 minutes. The cytoplasmic fraction is mixed with an equal volume of 0.2 M Tris-Hcl, pH 7.8, 25 mM EDTA, 0.3 M NaCl, 2% sodium dodecyl sulfate and 400 μg/ml of proteinase K, incubated for 1 hour at 37° C., then extracted once with an equal volume of phenol/cholorform (1:1/v:v) solution. Nucleic acids are obtained by ethanol precipitation of the separated aqueous phase. Total RNA is enriched by passage in 0.5 M NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% sarcosyl through an oligo dT(12-18) cellulose column. After washing, bound RNA is eluted in the same solution without sodium chloride.
EXAMPLE 3b Reverse Transcription of mRNA
Ten micrograms of 23.411 or HT 29 poly A+ RNA are primed for reverse transcription with oligo dT(12-18) and pdN6 primers. One hundred microliter reaction was performed for 4 hours at 42° C. with 200 units AMV reverse transcriptase (Life Science, Inc. St. Petersburg, Fla., U.S.A.). The RNA component of the cDNA/mRNA hybrids is replaced with the second complementary strand by treatment with RNase H, E. coli DNA polymerase I and E. coli DNA ligase at 12° C. and 22° C. for 1.5 hours each. Molecular ends are polished by treatment with T4 DNA polymerase. cDNA is phenol/cholorform extracted and purified over a “SEPHADEX G-50” spun column prepared in 10 mM Tris-HCl, pH 7.8, 1 mM EDTA (TE).
EXAMPLE 3c Cloning of cDNA for CEA-(g)
Synthetic DNA linkers
5′ pCCCGGG 3′
3′ GGGCCCTTAA 5′
are attached to the ends of cDNA by blunt end ligation with excess T4 DNA ligase. Excess linkers are removed by chromatography through “SEPHADEX G-50” (medium) in TE, and by fractionation on 0.8% low melting agarose gel. Based on Northern blot analysis of poly A+ RNA of the 23.411 and HT-29 cell lines, the size of the CEA-related mRNA is estimated at 1.7 kb. Therefore, cDNA fragments between 1 and 2 kb are recovered from gel slices and fragments are ethanol precipitated. After resuspension of cDNA in TE, EcoRI-cleaved lambda gt10 arms are added to cDNA at an estimated molar ration of 1:1. Ligation proceeds at 7° C. for 2 days in the presence of T4 DNA ligase. Aliquots of the ligation reaction are added to commercially-obtained packaging mix (Stratagene, San Diego, Calif., U.S.A.). Phage particles are obtained after in vitro packaging and infection of E. coli host NM514.
EXAMPLE 3d Screening of Recombinant Library
Five hundred thousand to one million packaged lambda particles are plated on lawns of E. coli NM514 and replicate patterns are lifted onto nitrocellulose sheets as described by W. D. Benton and R. W. Davis, Science, 196, 180-182, (1977). Positive phage are selected by hybridization with 32P-labeled LV7 cDNA insert probe that contained a domain repeated amoung various CEA family members. By this selection method, positive phage are obtained after multiple rounds of screening. Phage form individual plagues are amplified and titered, and these are used to prepare small quantities of recombinant phage DNA.
EXAMPLE 3e DNA Manipulation
Phage DNA is prepared according to T. Maniatis, E. Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, (1982). DNA segments are isolated from low melting agarose gels and inserted for subcloning into Bluescript plasmid vectors (Stratagene, San Diego, Calif., U.S.A.). DNA sequencing is performed by the didexoy termination method of F. Sanger, S. Nicklen and A. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74, 5463-5467, (1977). The nucleotide and translated sequence for a cDNA coding for CEA-(g) is given hereinabove (TM-4 (CEA-(g)).
EXAMPLE 4 Screening of a KG-1 cDNA Library with 32P-labelled CEA Probe, LV7 (CEA-(A))
A segment of cDNA coding for a portion of carcinoembroynic antigen [LV7 or CEA-(a)] was radiolabelled by random priming and used to detect homologous sequences on filter replicas of a commercial cDNA library prepared from KG-1 cells in bacteriophage vector λ gt11 (Clontech Laboratories, Inc., Palo Alto, Calif., U.S.A.). Hybridizations were performed at 68° C. in 2×SSSPE (1×SSPE—0.15 M NaCl, 0.01 M sodium phosphate and 1 mM EDTA, pH 7), 5×Denhart's solution and 100 μg of denatured salmon sperm DNA per ml, and post-hybridization washes were in 0.2×SSC, 0.25% sodium dodecyl sulfate.
Positive phage were picked, rescreened to homogeneity, and amplified for production of DNA. cDNA inserts were excised from phage DNA with EcoRI endonuclease and subcloned into the EcoRI site of the plasmid vector pBluescript KS. DNA sequencing on double-stranded DNA was by the method of Sanger et al, supra. The sequences of two different inserts from the KG-1 cDNA library are shown below:
pcKGCEA1:
1 acagcacagctgacagccgtactcaggaagcttctggatcctaggcttatctccacagag 60
61 gagaacacacaagcagcagagaccatggggcccctctcagcccctccctgcacacacctc 120
                        MetGlyProLeuSerAlaProProCysThrHisLeu
121 atcacttggaagggggtcctgctcacagcatcacttttaaacttctggaatccgcccaca 180
IleThrTrpLysGlyValLeuLeuThrAlaSerLeuLeuAsnPheTrpAsnProProThr
181 actgcccaagtcacgattgaagcccagccacccaaagtttctgaggggaaggatgttctt 240
ThrAlaGlnValThrIleGluAlaGlnProProLysValSerGluGlyLysAspValLeu
241 ctacttgtccacaatttgccccagaatcttgctggctacatttggtacaaagggcaaatg 300
LeuLeuValHisAsnLeuProGlnAsnLeuAlaGlyTyrIleTrpTyrLysGlyGlnMet
301 acatacgtctaccattacattacatcatatgtagtagacggtcaaagaattatatatggg 360
ThrTyrValTyrHisTyrIleThrSerTyrValValAspGlyGlnArgIleIleTyrGly
361 cctgcatacagtggaagagaaagagtatattccaatgcatccctgctgatccagaatgtc 420
ProAlaTyrSerGlyArgGluArgValTyrSerAsnAlaSerLeuLeuIleGlnAsnVal
421 acgcaggaggatgcaggatcctacaccttacacatcataaagcgacgcgatgggactgga 480
ThrGlnGluAspAlaGlySerTyrThrLeuHisIleIleLysArgArgAspGlyThrGly
481 ggagtaactggacatttcaccttcaccttacacctggagactcccaagccctccatctcc 540
GlyValThrGlyHisPheThrPheThrLeuHisLeuGluThrProLysProSerIleSer
541 agcagcaacttaaatcccagggaggccatggaggctgtgatcttaacctgtgatcctgcg 600
SerSerAsnLeuAsnProArgGluAlaIleGluAlaValIleLeuThrCysAspProAla
601 actccagccgcaagctaccagtggtggatgaatggtcagagcctccctatgactcacagg 660
ThrProAlaAlaSerTyrGlnTrpTrpMetAsnGlyGlnSerLeuProMetThrHisArg
661 ttgcagctgtccaaaaccaacaggaccctctttatatttggtgtcacaaagtatattgca 720
LeuGlnLeuSerLysThrAsnArgThrLeuPheIlePheGlyValThrLysTyrIleAla
721 ggaccctatgaatgtgaaatacggaacccagtgagtgccagccgcagtgacccagtcacc 780
GlyProTyrGluCysGluIleArgAsnProValSerAlaSerArgSerAspProValThr
781 ctgaatctcctcccaaagctgtccaagccctacatcacaatcaacaacttaaaccccaga 840
LeuAsnLeuLeuProLysLeuSerLysProTyrIleThrIleAsnAsnLeuAsnProArg
841 gagaataaggatgtcttaaccttcacctgtgaacctaagagtgaqaactacacctacatt 900
GluAsnLysAspValLeuThrPheThrCysGluProLysSerGluAsnTyrThrTyrIle
901 tggtggctaaatggtcagagcctccctgtcagtcccagggtaaagcgacccattgaaaac 960
TrpTrpLeuAsnGlyGlnSerLeuProValSerProArgValLysArgProIleGluAsn
961 aggatcctcattctacccaatgtcacgagaaatgaaacaggaccttatcaatgtgaaata 1020
ArgIleLeuIleLeuProAsnValThrArgAsnGluThrGlyProTyrGlnCysGluIle
1021 cgggaccgatatggtggcatccgcagtgacccagtcaccctgaatgtcctctatggtcca 1080
ArgAspArgTyrGlyGlyIleArgSerAspProValThrLeuAsnValLeuTyrGlyPro
1081 gacctccccagcatttacccttcattcacctattaccgttcaggagaaaacctctacttt 1140
AspLeuProSerIleTyrProSerPheThrTyrTyrArgSerGlyGluAsnLeuTyrPhe
1141 tcctgcttcggtgagtctaacccacgggcacaatattcttggacaattaatgggaagttt 1200
SerCysPheGlyGluSerAsnProArgAlaGlnTyrSerTrpThrIleAsnGlyLysPhe
1201 cagctatcaggacaaaagctctctatcccccaaataactacaaagcatagtgggctctat 1260
GlnLeuSerGlyGlnLysLeuSerIleProGlnIleThrThrLysHisSerGlyLeuTyr
1261 gcttgctctgttcgtaactcagccactggcaaggaaagctccaaatccatcacagtcaaa 1320
AlaCysSerValArgAsnSerAlaThrGlyLysGluSerSerLysSerIleThrValLys
1321 gtctctgactggatattaccctgaattctactagttcctccaattccattttctcccatg 1380
ValSerAspTrpIleLeuProEnd
1381 gaatcacgaagagcaagacccactctgttccagaagccctataatctggaggtggacaac 1440
1441 tcgatgtaaatttcatgggaaaacccttgtacctgacatgtgagccactcagaactcacc 1500
1501 aaaatgttcgacaccataacaacagctactcaaactgtaaaccaggataagaagttgatg 1560
1561 acttcacactgtggacagtttttcaaagatgtcataacaagactccccatcatgacaagg 1620
1621 ctccaccctctactgtctgctcatgcctgcctctttcacttggcaggataatgcagtcat 1680
1681 tagaatttcacatgtagtagcttctgagggtaacaacagagtgtcagatatgtcatctca 1740
1741 acctcaaacttttacgtaacatctcagggaaatgtggctctctccatcttgcatacaggg 1800
1801 ctcccaatagaaatgaacacagagatattgcctgtgtgtttgcagagaagatggtttcta 1860
1861 taaagagtaggaaagctgaaattatagtagagtctcctttaaatgcacattgtgtggatg 1920
1921 gctctcaccatttcctaagagatacagtgtaaaaacgtgacagtaatactgattctagca 1980
1981 gaataaacatgtaccacatttgcaaaaaa 2010
pcKGCEA2:
1 gggtggatcctaggctcatctccataggggagaacacacatacagcagagaccatggga 59
                                                     MetGly
60 cccctctcagcccctccctgcactcagcacatcacctggaaggggctcctgctcacagca 119
ProLeuSerAlaProProCysThrGlnHisIleThrTrpLysGlyLeuLeuLeuThrAla
120 tcacttttaaacttctggaacctgcccaccactgcccaagtaataattgaagcccagcca 179
SerLeuLeuAsnPheTrpAsnLeuProThrThrAlaGlnValIleIleGluAlaGlnPro
180 cccaaagtttctgaggggaaggatgttcttctacttgtccacaatttgccccagaatctt 239
ProLysValSerGluGlyLysAspValLeuLeuLeuValHisAsnLeuProGlnAsnLeu
240 actggctacatctggtacaaagggcaaatgacggacctctaccattacattacatcatat 299
ThrGlyTyrIleTrpTyrLysGlyGlnMetThrAspLeuTyrHisTyrIleThrSerTyr
300 gtagtagacggtcaaattatatatgggcctgcctacagtggacgagaaacagtatattcc 359
ValValAspGlyGlnIleIleTyrGlyProAlaTyrSerGlyArgGluThrValTyrSer
360 aatgcatccctgctgatccagaatgtcacacaggaggatgcaggatcctacaccttacac 419
AsnAlaSerLeuLeuIleGlnAsnValThrGlnGluAspAlaGlySerTyrThrLeuHis
420 atcataaagcgaggcgatgggactggaggagtaactggatatttcactgtcaccttatac 479
IleIleLysArgGlyAspGlyThrGlyGlyValThrGlyTyrPheThrValThrLeuTyr
480 tcggagactcccaagcgctccatctccagcagcaacttaaaccccagggaggtcatggag 539
SerGluThrProLysArgSerIleSerSerSerAsnLeuAsnProArgGluValMetGlu
540 gctgtgcgcttaatctgtgatcctgagactccggatgcaagctacctgtggttgctgaat 599
AlaValArgLeuIleCysAspProGluThrProAspAlaSerTyrLeuTrpLeuLeuAsn
600 ggtcagaacctccctatgactcacaggttgcagctgtccaaaaccaacaggaccctctat 659
GlyGlnAsnLeuProMetThrHisArgLeuGlnLeuSerLysThrAsnArgThrLeuTyr
660 ctatttggtgtcacaaagtatattgcagggccctatgaatgtgaaatacggaggggagtg 719
LeuPheGlyValThrLysTyrIleAlaGlyProTyrGluCysGluIleArgArgGlyVal
720 agtgccagccgcagtgacccagtcaccctgaatctcctcccgaagctgcccatgccttac 779
SerAlaSerArgSerAspProValThrLeuAsnLeuLeuProLysLeuProMetProTyr
780 atcaccatcaacaacttaaaccccagggagaagaaggatgtgttagccttcacctgtgaa 839
I1eThrIleAsnAsnLeuAsnProArgG1uLysLysAspValLeuAlaPheThrCysGlu
840 cctaagagtcggaactacacctacatttggtggctaaatggtcagagcctcccggtcagt 899
ProLysSerArgAsnTyrThrTyrIleTrpTrpLeuAsnGlyGlnSerLeuProValSer
900 ccgagggtaaagcgacccattgaaaacaggatactcattctacccagtgtcacgagaaat 959
ProArgValLysArgProIleGluAsnArgIleLeuIleLeuProSerValThrArgAsn
960 gaaacaggaccctatcaatgtgaaatacgggaccgatatggtggcatccgcagtaaccca 1019
GluThrGlyProTyrGlnCysGluIleArgAspArgTyrGlyGlyIleArgSerAsnPro
1020 gtcaccctgaatgtcctctatggtccagacctccccagaatttacccttacttcacctat 1079
ValThrLeuAsnValLeuTyrGlyProAspLeuProArgIleTyrProTyrPheThrTyr
1080 taccgttcaygagaaaacctcgacttgtcctgctttgcggactctaacccaccggcagag 1139
TyrArgSerGlyGluAsnLeuAspLeuSerCysPheAlaAspSerAsnProProAlaGlu
1140 tatttttggacaattaatgggaagtttcagctatcaggacaaaagctctttatcccccaa 1199
TyrPheTrpThrIleAsnGlyLysPheGlnLeuSerGlyGlnLysLeuPheIleProGln
1200 attactacaaatcatagcgggctctatycttgctctgttcgtaactcagccactggcaag 1259
IleThrThrAsnHisSerGlyLeuTyrAlaCysSerValArgAsnSerAlaThrGlyLys
1260 gaaatctccaaatccatgatagtcaaagtctctggtccctgccatggaaaccagacagag 1319
GluIleSerLysSerMetIleValLysValSerGlyProCysHisGlyAsnGlnThrGlu
1320 tctcattaatggctgccacaatagagacactgagaaaaagaacaggttgataccttcatg 1379
SerHisEnd
1380 aaattcaagacaaagaagaaaaaggctcaatgttattggactaaataatcaaaaggataa 1439
1440 tgttttcataatttttattggaaaatgtgctgattcttggaatgttttattctccagatt 1499
1500 tatgaactttttttcttcagcaattggtaaagtatacttttgtaaacaaaaattgaaaca 1559
1560 tttgcttttgctctctatctgagtgccccccc 1591 .
It will be appreciated that the instant specification and claims are set forth by way of illustration and not limitation and that various modifications and changes may be made without departing from the spirit and scope of the present invention.

Claims (2)

What is claimed is:
1. A polypeptide expressed by a cell that is transfected, infected or injected with a recombinant cloning vehicle, said recombinant cloning vehicle coding for a CEA family polypeptide selected from the group consisting of sequences TM-2, TM-3, KGCEA1 and KGCEA2, or a labeled form of said polypeptide.
2. A peptide having an amino acid sequence corresponding to the entire amino acid sequence or a fragment thereof of the expression product of a cell that is transfected, infected or injected with a recombinant cloning vehicle, said fragment exhibiting immunological cross-reactivity with a CEA family member and having no less than five amino acids, and said recombinant cloning vehicle coding for a CEA family polypeptide selected from the group consisting of sequences TM-2, TM-3 KGCEA1 and KGCEA2, or a synthetic peptide corresponding to said expression product, or a labeled form thereof.
US08/027,974 1986-08-13 1993-03-08 cDNAs coding for members of the carcinoembryonic antigen family Expired - Fee Related US6342583B1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US08/027,974 US6342583B1 (en) 1986-08-13 1993-03-08 cDNAs coding for members of the carcinoembryonic antigen family
US08/468,856 US6013772A (en) 1986-08-13 1995-06-06 Antibody preparations specifically binding to unique determinants of CEA antigens or fragments thereof and use of the antibody preparations in immunoassays
US08/468,859 US6022958A (en) 1986-08-13 1995-06-06 cDNAs coding for members of the carcinoembryonic antigen family

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US89636186A 1986-08-13 1986-08-13
US1668387A 1987-02-19 1987-02-19
US6003187A 1987-06-19 1987-06-19
US20767888A 1988-06-16 1988-06-16
US07/274,107 US5122599A (en) 1986-08-13 1988-11-21 CDNAS coding for members of the carcinoembryonic antigen family
US07/760,031 US5231009A (en) 1986-08-13 1991-09-13 Cdnas coding for members of the carcinoembryonic antigen family
US08/027,974 US6342583B1 (en) 1986-08-13 1993-03-08 cDNAs coding for members of the carcinoembryonic antigen family

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US76003192A Division 1986-08-13 1992-09-13

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US08/468,856 Division US6013772A (en) 1986-08-13 1995-06-06 Antibody preparations specifically binding to unique determinants of CEA antigens or fragments thereof and use of the antibody preparations in immunoassays

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US20070048860A1 (en) * 1997-10-10 2007-03-01 The Government Of The Usa, As Represented By The Secretary, Department Of Health And Human Services Carcinoembryonic antigen (CEA) peptides
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Cited By (4)

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Publication number Priority date Publication date Assignee Title
US20110123530A1 (en) * 2008-03-31 2011-05-26 Arron Joseph R Compositions and methods for treating and diagnosing asthma
US9684000B2 (en) 2010-12-16 2017-06-20 Genentech, Inc. Diagnosis and treatments relating to TH2 inhibition
US9995755B2 (en) 2010-12-16 2018-06-12 Genentech, Inc. Diagnosis and treatments relating to TH2 inhibition
US11226341B2 (en) 2010-12-16 2022-01-18 Genentech, Inc. Method of treating asthma using an IL-13 antibody

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